KR100657017B1 - L-FABP activator comprising Platycodon grandiflorum extract and its purification method - Google Patents
L-FABP activator comprising Platycodon grandiflorum extract and its purification method Download PDFInfo
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- KR100657017B1 KR100657017B1 KR1020040064128A KR20040064128A KR100657017B1 KR 100657017 B1 KR100657017 B1 KR 100657017B1 KR 1020040064128 A KR1020040064128 A KR 1020040064128A KR 20040064128 A KR20040064128 A KR 20040064128A KR 100657017 B1 KR100657017 B1 KR 100657017B1
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- KR
- South Korea
- Prior art keywords
- fabp
- methanol
- bellflower
- extract
- liver
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Abstract
본 발명은 도라지 추출물을 유효성분으로 함유하는 알코올성 지방간과 밀접한 관련이 있는 L-FABP(liver fatty acid binding protein) 증강제 및 그 정제방법에 관한 것이다.The present invention relates to an L-FABP (liver fatty acid binding protein) enhancer closely related to alcoholic fatty liver containing bellflower extract as an active ingredient and a method for purifying the same.
본 발명에 따른 L-FABP 증강제는 도라지의 메탄올 추출물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획한 후 1차, 2차 소수성 실리카겔 컬럼 크로마토그래피, 박층 크로마토그래피 및 HPLC 등 일련의 과정을 거쳐 정제되었으며 추출물, 정제단계의 물질, 정제물 등은 L-FABP를 증가시키게 되고 이로 인해 알코올로 인한 간의 지방축적이 감소됨으로써 지방간 예방용 기능성식품이나 약물에 유용하게 사용될 수 있게 되는 것이다.L-FABP enhancer according to the present invention was purified through a series of processes, such as primary, secondary hydrophobic silica gel column chromatography, thin layer chromatography and HPLC after fractionating methanol extract of bellflower in the order of nucleic acid, chloroform, ethyl acetate. , Substances in the refining stage, purified products, etc. increases the L-FABP, thereby reducing the fat accumulation of liver due to alcohol, which can be usefully used in functional foods or drugs for preventing fatty liver.
Description
도 1은 본 발명의 일 실시예로서 도라지로부터 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 bellflower as an embodiment of the present invention.
도 2는 도1의 각 단계 분획물의 L-FABP 증가활성을 실험한 결과를 나타낸 도표이다.Figure 2 is a chart showing the results of experiments to increase the L-FABP activity of each step fraction of FIG.
도 3은 본 발명의 일 실시예로서 분획된 메탄올 추출물로부터 정제단계를 보인 과정도이다.Figure 3 is a process showing a purification step from the fractionated methanol extract as an embodiment of the present invention.
도 4는 도 3의 분리물질의 정제도를 보인 크로마토그램이다.4 is a chromatogram showing the purification degree of the separation material of FIG.
본 발명은 도라지로부터 분리한 알코올로 인한 지방간 예방을 위한 간의 L-FABP (liver fatty acid binding protein) 증강제 및 그 정제방법에 관한 것이다.The present invention relates to a liver fatty acid binding protein (L-FABP) enhancer and a method for purifying the liver for preventing fatty liver due to alcohol isolated from bellflower.
최근 통계청 발표에 따르면 간질환에 의한 40대 사망자는 10만명당 41.4명으로 2위인 교통사고에 비해 38%나 높다. 여기에 알코올 소비량과 여성 음주인구의 급격한 증가 추세에 있는 우리나라의 알코올성 간질환 중 가장 많은 비율을 차지하는 지방간은 간질환 환자의 45%를 차지하고 있으며, 환자들은 대부분 이를 잘 인식하지 못하고 있다. 또한 지방간의 원인이 되는 비만, 지속적 음주, 지속적 약물 복용 등이 교정되지 않는다면 지방간염(steatsohepatitis), 간섬유화를 거쳐 간경변증으로 진행될 수 있다(참조 : Carol, A. C. et al., Alcoholism : Clinical and Experimental Research 25(5) : 49s (2001), Laurie, F. P., J Clin Pharmacol. 49 : 291 (2000), Charles, S. L., Clinica Chimica Acta. 257 : 59 (1997), Minoru, T. et al., J Clin Investigation 103 : 313 (1999)). 알코올의 장기 섭취는 지방산의 산화를 억제하고 에스테르화를 증가시켜 지방간을 유도하여 간 조직내의 콜레스테롤 대사, 단백질 합성 및 분비 등에 영향을 미쳐 간 기능 저하를 초래한다(참조 : Sugimoto, T. et al., J Hepatol. 36 : 157 (2002), Donohue, T. M. Jr. et al., Alcohol Clin. Exp. Res. 25 : 87S (2001)) 현재까지 정확히 지방간을 유발시키는 단일 인자(factor)는 밝혀져 있지 않으며 다양한 요인에 의하여 발병되는 것으로 알려져 있다. 알코올에 의한 간손상 관계를 규명하기 위한 대부분의 연구들은 흡수된 알코올의 90%를 분해하는 ADH(alcohol dehydrogenase) 및 MEOS(microsomal ethanol-oxidizing system)에 관한 것이다.According to a recent report by the National Statistical Office, the number of deaths in their 40s from liver disease is 41.4 per 100,000, 38% higher than the second-largest traffic accident. In addition, fatty liver, which occupies the largest proportion of alcoholic liver disease in Korea, which is rapidly increasing in alcohol consumption and female drinking population, accounts for 45% of patients with liver disease, and most patients are not aware of this. In addition, obesity, persistent drinking, and continuous medications that cause fatty liver can lead to liver cirrhosis through steatsohepatitis and liver fibrosis (Call, AC et al., Alcoholism: Clinical and Experimental Research). 25 (5): 49s (2001), Laurie, FP, J Clin Pharmacol. 49: 291 (2000), Charles, SL, Clinica Chimica Acta. 257 59 (1997), Minoru, T. et al., J Clin Investigation 103 : 313 (1999). Long-term intake of alcohol inhibits fatty acid oxidation and increases esterification, leading to fatty liver, which affects cholesterol metabolism, protein synthesis and secretion in liver tissue, resulting in decreased liver function (see Sugimoto, T. et al. , J Hepatol. 36: 157 (2002), Donohue, TM Jr. et al., Alcohol Clin. Exp. Res. 25: 87S (2001)). It is known to be caused by various factors. Most studies to determine the relationship between alcohol-induced liver damage are related to alcohol dehydrogenase (ADH) and microsomal ethanol-oxidizing system (MEOS), which break down 90% of absorbed alcohol.
장기간 알코올 섭취 후에 분해 대사물로 생성되는 acetealdehyde 및 acetate에 의한 지방대사의 이상 및 중성지방의 축적으로 발생되는 지방간과 이때 변화되는 L-FABP (liver fatty acid binding protein)와의 상호 연관 관계는 중요하다. 이는 알코올은 폐와 신장을 통하여 10% 정도가 체외로 배출되고 나머지는 체내에 흡수된 후 약 90%가 간에서 산화되므로 알코올 섭취에 의하여 여러 조직의 FABP중에서 특히 간세포의 L-FABP의 변화가 가장 클 것으로 예상되기 때문이다. 즉 지방산 합성은 간의 cytosol에서 일어나지만 지방산의 분해는 미토콘드리아(mitochondria) 및 퍼옥시좀(peroxisome)에서 일어나기 때문에 알코올에 의하여 L-FABP의 양과 활성이 감소 될 경우 이들 소기관으로 지방산 이동에 변화가 생길 것이며 이로 인한 지방산 분해감소가 일어나 결과적으로는 간에서의 중성지방의 합성 증가 및 혈중 중성지방이 증가될 것이다.Correlation between fatty liver resulting from abnormal metabolism of aceticaldehyde and acetate produced by degrading metabolites and accumulation of triglycerides after long-term alcohol intake and the altered fatty acid binding protein (L-FABP) is important. This is because alcohol is excreted through the lungs and kidneys by about 10% and the rest is absorbed into the body, and about 90% is oxidized in the liver. It is expected to be large. That is, fatty acid synthesis occurs in the cytosol of the liver, but decomposition of fatty acids occurs in mitochondria and peroxisome, so if the amount and activity of L-FABP is reduced by alcohol, fatty acid migration to these organelles will change. This will result in reduced fatty acid degradation resulting in an increase in the synthesis of triglycerides in the liver and an increase in triglycerides in the blood.
L-FABP는 간(liver)과 장(intestine)에 분포하며 Ockner에 의하여 1970년대 초에 연구가 시작되었다. 보통 간세포질에서는 전체 단백질 중의 3 ∼ 5% 정도를 차지하며(참조 : Robert, K. et al., The journal of Clinical Investigation, 54 : 326 (1974), Robrtt, K. et al., J. Clin. Invest. 64 : 172 (1979), Robert, K. et al., J. Clin. Invest. 65 : 1013 (1980)), 크기는 14.3 kDa이고 장쇄 지방산(long chain fatty acid)과의 높은 친화력을 갖고 있으며 외부로부터 유입되는 지방산을 세포내 소기관으로 운반하는 운반단백질(carrier protein)로 알려져 있다. 간의 FABP는 다른 조직에 분포하고 있는 일반적인 FABP계열과 다르게 여러 종류의 ligand와 결합할 수 있으며 한 번에 2 개의 ligand와 결합하는 특성을 갖고 있다(참조 : Mohinder, P. et al., J.B.C. 264 : 13780 (1989), Asim, K. et al., Nutritional Biochemistry 8 : 548 (1997), Judith, K. et al., Biochimica et Biophysica Acta. 1145 : 257 (1993), Alfred, E. A. et al., Biochem. J. 300 : 827 (1994)). 고지방식이나 고농도의 당을 함유한 식이를 장기간 투여하였을 경우 간, 심장, 소장, 지방조직 등의 FABP가 증가하였고, fibrate계열의 약품 같은 혈청 지질 강하제는 간 세포내에서 유리지방산의 흡수속도와 FABP의 양을 증가시킨다는 보고가 있다(참조 : Ngoc, V. D. et al., J.B.C., 273 : 25713 (1998), Poirier, H. et al., Biochem. 355 : 481 (2001), Guy, R. et al., Biochemical and Biophysical research communication. 80 : 327 (1978), Galli, A. et al., J.B.C. 276 : 68 (2001)). 또한 in vitro 실험에서 L-FABP는 지방대사 관여 효소의 활성에 영향을 미친다는 것이 보고되어 유리 지방산(free fatty acid)의 이용과 대사에 변화가 생겼을 경우, 조직에 존재하는 FABP의 양 또한 변화가 있으며 이 단백질이 세포내의 지방 이용에 중요한 역할을 한다는 것을 간접적으로 보여주고 있다. 현재 알려진 바로는 L-FABP의 발현은 성호르몬(참조 : Robert, K. et al., J. Clin. Invest. 64 : 172 (1979)), 알코올, peroxisome proliferators, 지방산 등에 영향을 받는 것으로 알려져 있으며(참조 : Maria, Y. et al., Biochemical and Biophysical research communications 71 : 809 (1976), John, R. et al., J.B.C. 266 : 5486 (1991)), 생체내 증가된 유리 지방산에 대한 효소 및 세포막 운반 과정(enzymatic and membrane transport process)에 있어 역작용(adverse effect)에 대한 방어 기작으로 L-FABP의 증가가 보고되고 있으며 이로 인하여 증가된 지방산은 미토콘드리아, 퍼옥시솜, 소포체(endoplasmic reticulum) 등으로 보내지며 이들 소기관에서 산화(β-oxidation)되거나 중성지방(triglyceride)의 형태로 저장된다.L-FABP is distributed in the liver and intestine and was first studied by Ockner in the early 1970s. Usually in the hepatoplasm, it accounts for about 3-5% of the total protein (Robert, K. et al., The journal of Clinical Investigation, 54: 326 (1974), Robrtt, K. et al., J. Clin). Invest. 64 : 172 (1979), Robert, K. et al., J. Clin. Invest. 65: 1013 (1980)), its size is 14.3 kDa, has a high affinity with long chain fatty acids, and is known as a carrier protein that transports fatty acids from the outside into intracellular organelles. Unlike the common FABP family distributed in other tissues, hepatic FABP can bind to several ligands and has the property of binding to two ligands at once (see Mohinder, P. et al., JBC 264: 13780 (1989), Asim, K. et al., Nutritional Biochemistry 8: 548 (1997), Judith, K. et al., Biochimica et Biophysica Acta. 1145: 257 (1993), Alfred, EA et al., Biochem J. 300: 827 (1994)). Long-term administration of high-fat diet or high-sugar diet increased FABP in liver, heart, small intestine, and adipose tissue. Serum lipid-lowering agents such as fibrate-based drugs absorbed free fatty acids and FABP in liver cells. It has been reported that the amount of is increased (Ngoc, VD et al., JBC, 273: 25713 (1998), Poirier, H. et al., Biochem. 355: 481 (2001), Guy, R. et al. ., Biochemical and Biophysical research communication.80: 327 (1978), Galli, A. et al., JBC 276: 68 (2001)). In vitro experiments have also reported that L-FABP affects the activity of enzymes involved in fat metabolism. Therefore, if there is a change in the use and metabolism of free fatty acids, the amount of FABP present in tissues also changes. Indirectly, the protein plays an important role in the use of fat in cells. It is now known that L-FABP expression is affected by sex hormones (Robert, K. et al., J. Clin. Invest. 64: 172 (1979)), alcohols, peroxisome proliferators, fatty acids, etc. (See Maria, Y. et al., Biochemical and Biophysical research communications 71: 809 (1976), John, R. et al., JBC 266: 5486 (1991)), enzymes for increased free fatty acids in vivo and Increases in L-FABP have been reported as a defense mechanism against the adverse effects in the enzymatic and membrane transport process, resulting in increased fatty acids such as mitochondria, peroxysomes, and endoplasmic reticulum. It is sent and stored in the form of β-oxidation or triglyceride in these organelles.
도라지(Platycodon grandiflorum)는 한국, 중국 동북부, 일본에 자생하며 햇빛이 잘 쪼이는 산야의 초원에서 자라는 다년초이다. 또한 초롱꽃과에 속하는 다년 생초로 뿌리는 굵고 줄기는 단생 또는 족생(簇生)하여 높이 40 ∼ 100 cm이다. 잎은 호생 또는 윤생하며 거의 잎자루 없이 긴 계란형 또는 타원형이다. 꽃은 6 ∼ 8 월에 피며 꽃색은 청자색으로 종모양이며 줄기의 끝이나 가지 끝에 하나씩 정생하여 핀다. 흰꽃이 피는 계통을 백도라지라고 하며 뿌리는 각혈성환자의 약제 또는 식용으로 쓰인다. 도라지의 뿌리는 식용채소 또는 약용으로 이용되어 왔으며, 뿌리를 물로 씻어 말리거나 껍질을 벗겨서 말리고 건조시킨 것을 길경근이라고 하며 약용한다. 뿌리에는 2%의 트리텔페노이드 사포닌(triterpenoid saponin) 및 0.03%의 스테롤(sterol)이 함유되어 있다(참조 : Saeki, T. et al., Planta Med. 65 : 428 (1937)). 그 외 당류로서는 이누린(inulin), 프라티코디닌(platycodinin : 과당 10 분자로 구성되는 다당류)이 함유되어 있다. 한방에서 도라지의 주요 효능은 기침, 가래 등과 폐의 장해와 밀접한 인후통, 실음(失音), 배뇨곤란, 설사, 후중(後重) 등의 증상에 좋은 것으로 알려져 있다(참조 : Lee, K. J. et al., Food Chem. Toxicol. 40 : 517 (2002), Han, S. B. et al., Int. Immunopharmacol. 1 : 1969 (2001), Choi, C. Y. et al., Cancer Lett. 166 : 17 (2001)). 생쥐에서 acetaminophen에 대한 간보호 효과(참조 : Lee K. J. et al., Cancer Lett. 174 : 73 (2001)) 및 랫트에서 고지방식이로 유도된 hyperlipidemia에 대한 효과(참조 : Kim, K. S. et al., J. Nutr. Sci. Vitaminol. 41 : 485 (1995)) 등이 입증되었으나, 알코올로 인한 지방간 예방을 위한 L-FABP 증강활성이 보고되어진 바는 없다.The bellflower ( Platycodon grandiflorum ) is a perennial herb that grows in the grassland of Sanya, native to Korea, northeastern China and Japan. In addition, it is a perennial plant belonging to the campanula, the roots are thick, and the stems are single or pedigerous, 40 to 100 cm high. The leaves are regenerated or resilient, long egg-shaped or oval with almost no petioles. Flowers bloom in June-August, flower color is blue purple, bell-shaped and blooms one by one at the end of stem or branch. The white flowering system is called Baekdoraji, and the root is used for medicine or food for keratinized patients. The root of bellflower has been used for edible vegetable or medicinal, and the root is washed with water, dried or peeled, dried and dried. The root contains 2% triterpenoid saponin and 0.03% sterol (Saeki, T. et al., Planta Med. 65: 428 (1937)). Other saccharides include inulin and praticindin (polysaccharides composed of 10 fructose). It is known that the main effect of bellflower in oriental medicine is good for symptoms such as sore throat, nausea, difficulty urination, diarrhea, and post-heavy weight, which are closely related to cough, phlegm, and lung disorders (Lee, KJ et al. , Food Chem. Toxicol. 40: 517 (2002), Han, SB et al., Int. Immunopharmacol. 1: 1969 (2001), Choi, CY et al., Cancer Lett. 166: 17 (2001)). Hepatoprotective effect on acetaminophen in mice (Lee KJ et al., Cancer Lett. 174: 73 (2001)) and on high-fat diet-induced hyperlipidemia in rats (Kim, KS et al., J. Nutr. Sci. Vitaminol. 41: 485 (1995)) has been demonstrated, but L-FABP potentiating activity for preventing fatty liver due to alcohol has not been reported.
따라서 본 발명자들은 상기와 같이 간에서 L-FABP 증강제로 사용할 수 있도록 기능성식품, 식품의약, 의약의 개발에 있어 요구되는 식품신소재를 개발함에 있 어 안전성을 확보하고, 물질의 임상에 대한 위험성을 감소시키기 위해 오랫동안 식품으로 이용해 온 200여 종의 식·약용식물을 대상으로 L-FABP 증강물질을 검색하였다. 검색을 수행하던 중 국내에서 오랫동안 상식해 온 도라지의 메탄올 추출물에 높은 L-FABP 증가활성을 확인하고 메탄올 추출물로부터 상기 활성을 갖는 저분자 물질을 정제함으로써 본 발명을 이루게 되었다. Therefore, the present inventors secure safety in developing new food materials required for the development of functional foods, food medicines, and medicines so that they can be used as L-FABP enhancers in the liver as described above, and reduce the risk of the substance to clinical In order to find out about 200 food and medicinal plants that have been used for a long time to search for L-FABP enhancer. During the search, the present invention was achieved by confirming the high L-FABP increasing activity in methanol extract of bellflower, which has long been common sense in Korea, and purifying the low molecular weight substance having the above activity from methanol extract.
본 발명의 목적은 상기와 같은 종래의 문제점을 해소하기 위한 것으로, 도라지 추출물을 유효성분으로 함유하는 간에서 지방축적을 저해하는 L-FABP 증강제를 제공함을 목적으로 한다.An object of the present invention is to solve the conventional problems as described above, and to provide an L-FABP enhancer that inhibits fat accumulation in the liver containing bellflower extract as an active ingredient.
또한 본 발명의 또 다른 목적은 도라지로부터 L-FABP 증강제를 분리하는 방법을 제공함에 있다.It is another object of the present invention to provide a method for separating L-FABP enhancer from bellflower.
또한 본 발명의 또 다른 목적은 본 발명의 L-FABP 증강제를 함유하는 알코올성 지방간의 예방, 치료용 기능성 식품이나 약학적 조성물을 제공함에 있다.Another object of the present invention is to provide a functional food or pharmaceutical composition for preventing or treating alcoholic fatty liver containing the L-FABP enhancer of the present invention.
상기와 같은 목적을 달성하기 위하여, 본 발명은 도라지(Platycodon grandiflorum) 추출물을 유효성분으로 함유하는 간 L-FABP 증강제를 제공한다.In order to achieve the above object, the present invention provides a liver L-FABP enhancer containing a bellflower ( Platycodon grandiflorum ) extract as an active ingredient.
본 발명에 있어서, 도라지 추출물은 물, 친수성유기용매 또는 이들의 혼합용매로도 추출될 수 있으나, 바람직하게는 도라지의 메탄올 추출물인 것을 특징으로 한다. 여기서, 메탄올은 50%~100%로 물에 희석하여 사용할 수도 있으나, 바람직하게는 100% 메탄올로 추출하는 것이 활성성분의 용출에 유리하다.In the present invention, the bellflower extract may be extracted with water, a hydrophilic organic solvent or a mixed solvent thereof, but is preferably a methanol extract of bellflower. Here, methanol may be used diluted with water at 50% to 100%, but preferably extracted with 100% methanol to elute the active ingredient.
본 발명에 있어서, 도라지 추출물은 도라지를 메탄올로 추출한 후 각종 유기용매로 분획할 수 있는데, 바람직하게는 도라지를 분쇄한 후 실온에서 메탄올로 추출하고 그 농축물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획하고 남은 것을 메탄올에 녹여 각각을 여과포로 여과하고 잔사를 감압 농축한 것을 특징으로 한다. 여기서, 핵산, 클로로포름, 에틸아세테이트는 순차적으로 일부 또는 전체 분획할 수 있다.In the present invention, the bellflower extract may be fractionated with various organic solvents after extracting the bellflower with methanol. Preferably, the bellflower is pulverized and extracted with methanol at room temperature and the concentrate is fractionated in the order of nucleic acid, chloroform and ethyl acetate. The residue was dissolved in methanol, and each was filtered with a filter cloth, and the residue was concentrated under reduced pressure. Here, the nucleic acid, chloroform, ethyl acetate may be partially or completely fractionated sequentially.
본 발명에 있어서, 도라지 추출물은 각종 유기용매로 분획한 후 각종 크로마토그래피로 정제될 수 있는데, 바람직하게는 도라지의 메탄올 추출물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획한 후, 소수성 실리카겔 컬럼 크로마토그래피, 박층(Thin-Layer) 크로마토그래피 및 HPLC(High Performance Liquid Chromatography)의 일련의 과정을 거쳐 정제된 것을 특징으로 한다. 구체적으로 본 발명은 크로마토그래피를 위해 1차, 2차 실리카겔 60 컬럼 크로마토그래피, ODS gel plate로 Prep-TLC 및 HPLC(u-Bondapak C18)를 사용하였다. 여기서, 크로마토그래피는 순차적으로 일부 또는 전체 정제할 수 있다.In the present invention, the bellflower extract may be purified by various chromatography after fractionation with various organic solvents. Preferably, the methanol extract of bellflower is fractionated in the order of nucleic acid, chloroform, ethyl acetate, hydrophobic silica gel column chromatography, It is characterized by being purified through a series of thin-layer chromatography and HPLC (High Performance Liquid Chromatography). Specifically, the present invention used prep-TLC and HPLC (u-Bondapak C 18 ) as a primary, secondary silica gel 60 column chromatography, ODS gel plate for chromatography. Here, the chromatography may be partially or fully purified sequentially.
본 발명에 있어서, 상기 간 L-FABP 증강제는 알코올성 지방간의 예방 및 치료에 사용되는 것을 특징으로 한다. 알코올성 지방간 치료제인 Fibrate 계열의 혈청지질강하약제에 의한 L-FABP의 양 증가 효과가 당업계에 잘 알려져 있으므로(Poirier, H. et al., Biochem. 355 : 481 (2001); Guy, R. et al., Biochemical and Biophysical research communication. 80 : 327 (1978); Tsutsumi. M. et al., Alcohol Clin. Exp. Res. 25(6 Suppl) : 75S (2001)), 본 발명은 알코올성 지방간 예방 및 치료에 있어 L-FABP의 활성을 증가시킴으로써 중성지방의 합성 및 축적을 억제한다는 관점에서 기존의 지방간 질환 약제보다 유용성이 높을 것이다. 또한, 본 발명은 식용식물인 도라지로부터 분리한 천연물질로 기존의 유기합성약제에 비해 부작용이 없고, 안전성이 매우 높다.In the present invention, the liver L-FABP enhancer is used for the prevention and treatment of alcoholic fatty liver. Since the effect of increasing the amount of L-FABP by Fibrate-based serum lipid-lowering drugs of alcoholic fatty liver drugs is well known in the art (Poirier, H. et al., Biochem. 355: 481 (2001); Guy, R. et. al., Biochemical and Biophysical research communication.80: 327 (1978); Tsutsumi.M. et al., Alcohol Clin.Exp. Res. 25 (6 Suppl): 75S (2001)). In terms of the treatment of the synthesis and accumulation of triglycerides by increasing the activity of L-FABP in the treatment will be more useful than conventional fatty liver disease drugs. In addition, the present invention is a natural material separated from bellflower, which is an edible plant, has no side effects and is very safe compared to conventional organic synthetic pharmaceuticals.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 도라지의 메탄올 추출물을 핵산, 클로로포름, 에틸아세테이트, 메탄올로 분획하는 단계와; 상기 단계에서 획득한 메탄올 분획물을 실리카겔 60G 컬럼에 주입한 다음 클로로포롬 : 메탄올 = 60 : 40으로 용출분획하여 수득하는 단계와; 상기 수득공정에서의 활성분획물을 ODS gel plate와 메탄올 : 물 = 7 : 3의 전개제로 분획하는 단계와; 상기의 활성 분획을 C18 Symmetry column이 부착된 HPLC를 사용하여 물과 메탄올의 gradient로 용출분획함으로써 L-FABP증강물질을 분리하는 도라지로부터 분리한 L-FABP 증강제의 정제방법을 제공한다.In order to achieve another object of the present invention, the present invention comprises the steps of fractionating the methanol extract of bellflower into nucleic acid, chloroform, ethyl acetate, methanol; Injecting the methanol fraction obtained in the above step into a silica gel 60G column and then eluting with chloroform: methanol = 60: 40; Fractionating the active fraction in the obtaining step with a developer of an ODS gel plate and methanol: water = 7: 3; The active fraction is eluted with a gradient of water and methanol using HPLC with a C 18 symmetry column to provide a method for purifying L-FABP enhancer isolated from bellflower for separating L-FABP enhancer.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 본 발명에 따른 간 L-FABP 증강제를 유효성분으로 함유하는 알코올성 지방간의 예방 및 치료용 기능성식품을 제공한다.In order to achieve another object of the present invention, the present invention provides a functional food for preventing and treating alcoholic fatty liver containing the liver L-FABP enhancer according to the present invention as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 상기 본 발명에 따른 간 L-FABP 증강제를 유효성분으로 함유하는 알코올성 지방간의 예방 및 치료용 약학적 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for the prevention and treatment of alcoholic fatty liver containing the liver L-FABP enhancer according to the present invention as an active ingredient.
이하 상기와 같이 구성된 본 발명의 도라지로부터 분리한 간에서의 L-FABP 증강물질 및 그 정제방법의 기술적 사항을 좀더 상세하게 설명하면 다음과 같다. Hereinafter, the technical details of the L-FABP enhancer in the liver separated from the bellflower of the present invention configured as described above and its purification method will be described in more detail.
본 발명에 있어서, 도라지 추출물에는, 추출, 분획 및 정제 처리의 각 단계에서 얻어지는 모든 추출액, 분획 및 정제물, 그 희석액 또는 농축액, 또는 그 건조물 중 어느 하나도 포함하는 것으로 한다.In the present invention, the bellflower extract includes all of the extracts, fractions and purified products obtained in each step of the extraction, fractionation and purification treatment, diluents or concentrates thereof, or dried products thereof.
본 발명에 있어서, 상기 추출용매로서 사용할 수 있는 친수성유기용매로는, 예컨대, 메탄올, 에탄올, 프로필알콜, 이소프로필알콜 등의 탄소수1~5의 저급알콜;아세톤, 메틸에틸케톤 등의 저급지방족케톤; 1,3-부틸렌글리콜, 프로필렌글리콜, 글리세린 등의 탄소수 2~5의 다가알콜 등을 들 수 있고, 이들 친수성 유기용매와 물과의 혼합용액 등을 이용할 수 있다.In the present invention, as the hydrophilic organic solvent that can be used as the extraction solvent, for example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone ; C2-C5 polyhydric alcohols, such as 1, 3- butylene glycol, a propylene glycol, and glycerin, etc. are mentioned, The mixed solution of these hydrophilic organic solvents, and water can be used.
구체적으로는, 본 발명자들은 200여 종의 식·약용식물을 대상으로 추출한 추출물 중에서 도라지의 메탄올 추출물이 비교적 높은 L-FABP 증가활성을 가지고 있음을 발견하였다. 이에 도라지에 포함된 L-FABP 증강물질을 분리하고자 실온에서 100% 메탄올로 추출한 후 이 추출물을 핵산, 클로로포름, 에틸아세테이트 순으로 분획을 실시한 다음, L-FABP 증가활성이 높았던 메탄올 분획물을 실리카겔 컬럼 크로마토그래피하였다. 그 결과 L-FABP 증가활성이 높은 분획을 모아 ODS gel plate로 Prep-TLC를 실시한 후 높은 활성의 분획을 HPLC를 실시하여 정제분획을 얻었다.Specifically, the present inventors found that methanol extract of bellflower has a relatively high L-FABP increasing activity among extracts extracted from 200 food and medicinal plants. The L-FABP enhancer contained in the bellflower was extracted with 100% methanol at room temperature to separate the extract, followed by fractionation of the extract in the order of nucleic acid, chloroform, ethyl acetate, and then methanol fractions having high L-FABP increasing activity were purified by silica gel column chromatography. It was done. As a result, the fractions with high L-FABP increasing activity were collected and prep-TLC was performed on the ODS gel plate, and the highly active fractions were purified by HPLC.
본 발명의 원료인 도라지 추출물은 당업계에 알려진 통상의 방법에 의해 과립, 정제, 캡슐 또는 드링크제 등의 형태로 제제화될 수 있다. 또한, 보존이나 취급을 용이하게 하기 위하여 덱스트린, 사이클로덱스트린 등의 통상 제제화에 사용 되는 캐리어, 그 밖의 임의의 조제를 부가하여도 좋다. 또한, 식품 또는 약물에 통상적으로 첨가되는 보조적인 원료 또는 첨가물로는 특히 제한되지 않으나, 예컨대, 포도당, 과당, 자당, 말토오스, 솔비톨, 스테비오사이드, 롭소사이드, 콘시럽, 유당, 구연산, 주석산, 사과산, 호박산, 유산, L-아스코르빈산, d1-α-토코페롤, 엘리솔빈산 나트륨, 글리세린, 프로필렌글리콜, 글리세린지방산 에스테르, 폴리글리세린지방산에스테르, 자당지방산에스테르, 솔비탄지방산에스테르, 아라비아껌, 칼라기난, 카제인, 젤라틴, 펙틴, 한천, 비타민 B류, 니코틴산 아미드, 팬트텐산 칼슘, 아미노산류, 칼슘염류, 색소, 향료, 보존제 등을 들 수 있다.Bellflower extract, which is a raw material of the present invention, may be formulated in the form of granules, tablets, capsules, or drinks by conventional methods known in the art. In addition, in order to facilitate preservation and handling, a carrier used for the usual formulation of dextrin, cyclodextrin and the like, and other optional preparations may be added. In addition, auxiliary raw materials or additives conventionally added to foods or drugs are not particularly limited, for example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, loxoside, corn syrup, lactose, citric acid, tartaric acid, malic acid , Succinic acid, lactic acid, L-ascorbic acid, d1-α-tocopherol, sodium elisolvinate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic , Casein, gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium panthenate, amino acids, calcium salts, pigments, flavors, preservatives and the like.
본 발명의 도라지 추출물은, 이를 그대로 음식물중에 첨가하여 알코올성 지방간 예방 및 치료용의 음식물로 할 수 있다. 이와 같이 하여 얻어지는 본 발명의 음식물은, 일상적으로 섭취하는 것이 가능하기 때문에, 높은 간 기능 개선 효과를 기대할 수 있어, 매우 유용하다.Bellflower extract of the present invention can be added to the food as it is to food for preventing and treating alcoholic fatty liver. Since the food and drink of the present invention thus obtained can be consumed on a daily basis, a high liver function improvement effect can be expected and is very useful.
이와 같은 음식물에 있어서의 도라지 추출물의 첨가량은, 대상인 음식물의 종류에 따라 달라 일률적으로 규정할 수 없지만, 음식물 본래의 맛을 손상시키지 않는 범위에서 첨가하면 되며, 대상음식물에 대하여 통상 0.01~50질량%, 바람직하기로는 0.1~20질량%의 범위이다. 또한, 과립, 정제 또는 캡슐형태의 음식물의 경우에는 통상 0.1~100질량%, 바람직하기로는 5~100질량%의 범위에서 첨가하면 된다.The addition amount of bellflower extract in such a food cannot be uniformly prescribed | regulated depending on the kind of food to which it is intended, but what is necessary is just to add it in the range which does not impair the original taste of food, Usually 0.01-50 mass% with respect to the food of interest. Preferably, it is the range of 0.1-20 mass%. In the case of food in the form of granules, tablets or capsules, it is usually 0.1 to 100% by mass, preferably added in the range of 5 to 100% by mass.
또한, 본 발명의 간 L-FABP 증강제의 유효성분인 도라지 추출물은, 성인 하루당 섭취량이 1~3000mg이 되도록 투여하는 것이 적당하다. 또한, 투여량은 연령, 증상 등에 따라 적당히 증감하는 것이 가능하다.In addition, the bellflower extract, which is an active ingredient of the liver L-FABP enhancer of the present invention, is appropriately administered so that the daily intake amount of adult is 1 ~ 3000mg. In addition, the dosage can be appropriately increased or decreased depending on age, symptoms, and the like.
이하, 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 당업계에서 통상의 지식을 가진 자에게 있어서는 자명할 것이다.Hereinafter, the present invention configured as described above will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, 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 : 도라지로부터 간내 L-FABP 증강물질 분리를 위한 추출조건 선정과 증강활성능 확인Example 1 Selection of Extraction Conditions and Confirmation of Enhancing Activity for Separation of L-FABP Enhancers from Liver
200여 종의 식·약용식물에서 냉수, 열수, 메탄올로 추출한 추출물을 검색하던 중 도라지의 메탄올 추출물이 L-FABP 증강활성능을 나타내는 것을 확인하였다. 이에 도라지에 포함된 L-FABP 증강물질을 분리하고자 도라지를 100℃에서 5분간 blanching하여 생체세포내 효소를 불활성화시키고 분쇄한 후, 실온에서 시료의 다섯배의 100% 메탄올로 24시간 정치하여 추출하는 방법을 3회 반복한 다음, 그 추출물을 감압 농축하였다(C-M). 상기의 농축물에 1L의 핵산, 클로로포름, 에틸아세테이트 순으로, 각 용매당 36시간 동안 3회 용매를 교환하여 분획깔대기로 분획하고(M-H, M-C, M-E) 남은 것을 메탄올에 녹였으며(M-M) 각각의 용매 분획물을 여과포로 여과하고 여액을 감압 농축한 후 건조하여 추출물을 얻었다. 도 1은 본 발명의 일 실시예로서 도라지로부터 메탄올추출 및 추출물의 유기용매 분획 단계를 보인 과정도이다.Searching for extracts extracted with cold water, hot water and methanol from 200 food and medicinal plants, it was confirmed that the methanol extract of bellflower showed L-FABP enhancement activity. In order to separate the L-FABP enhancer contained in the bellflower, blanching the bellflower at 100 ° C. for 5 minutes to inactivate and pulverize enzymes in biological cells, and then extract it by standing with five
각 분획물에 대한 L-FABP 증가활성 측정 실험을 하기 위해 ATCC에서 분양받은 HepG2 cell(HB-8065)에 CYP2E1을 transfection한 HepG2 2E1 LNCX2 cell line (한양대 박장환 교수 분양)을 48시간 동안 2% 알코올과 같이 배양한 다음 lysis buffer를 이용하여 얻은 cell cytosol을 취하여 ELISA 방법을 이용하여 다음과 같이 측정하였다. Immuno-well plate에 glutaraldehyde를 100 ㎕ (10 ㎕ /ml D.W)씩 well에 분주하여 37℃, 1시간 반응시킨 후 증류수로 well plate를 세척하고 물기를 제거하였다. 상기의 cytosol 상등액을 100 ㎕ 분주하고 37℃, 1시간 반응시킨 후 L-FABP antibody(mouse)(Hycult biotechnology, Uden, Netherland)를 1:4,000으로 blocking solution(0.5% BSA)에 희석하여 100 ㎕ 씩 분주하고 37℃, 1시간 반응시켰다. Anti-mouse IgG conjugated peroxidase(Sigma, St. Louis, USA)를 1:4,000으로 blocking solution에 희석하여 37℃, 1시간 반응시킨 후 기질[TMB 10 mg/mL DMSO, 3% H2O2, 50 mM sodium acetate buffer(pH 5.1)]을 well당 200 ㎕ 씩 넣어 15 분간 빛을 차단한 채 반응시켰다. 1 M H2SO4 100 ㎕를 첨가하여 반응을 완전히 중지시킨 후 micro plate reader(Model 550, BIO-RAD Laboratories, USA)을 사용하여 450 nm에서 흡광도를 측정하였다. 시료에 의한 L-FABP양의 증가활성은 아래 식에 따라 환산하였다.In order to measure L-FABP increase activity for each fraction, the HepG2 2E1 LNCX2 cell line (Professor Hanyang University Park Jang Hwan) transfected with CYP2E1 in ATCC-prepared HepG2 cells (HB-8065) was treated with 2% alcohol for 48 hours. After culturing, the cell cytosol obtained using the lysis buffer was taken and measured using the ELISA method as follows. 100 μl (10 μl / ml DW) of glutaraldehyde was dispensed into the wells in an immuno-well plate and allowed to react at 37 ° C. for 1 hour, followed by washing the well plate with distilled water and removing water. Dispense 100 μl of the cytosol supernatant, react at 37 ° C for 1 hour, and dilute L-FABP antibody (mouse) (Hycult biotechnology, Uden, Netherland) with a blocking solution (0.5% BSA) of 1: 4,000 (100 μl). Aliquoting was performed and reaction was carried out at 37 ° C for 1 hour. Anti-mouse IgG conjugated peroxidase (Sigma, St. Louis, USA) diluted 1: 4,000 in a blocking solution and reacted for 1 hour at 37 ℃, substrate [
L-FABP 증가활성(%) = [1 - (As - Ab / Ac - Ab) ] × 100 % Increase in L-FABP = [1-(As-Ab / Ac-Ab)] × 100
Ac : 2% 알코올을 처리한 control cell lysate의 흡광도 Ac: absorbance of control cell lysate treated with 2% alcohol
Ab : ELISA 측정시 증류수만을 처리한 well의 흡광도 Ab: absorbance of wells treated only with distilled water for ELISA measurement
As : 2% 알코올 및 시료를 처리한 cell lysate의 흡광도 As: absorbance of cell lysate treated with 2% alcohol and sample
상기와 같이 분획에 따른 L-FABP 증가활성을 측정한 결과, 처음 메탄올 추출물(C-M)에서도 상당한 활성을 나타내나, 최종 메탄올 분획(M-M)에서 가장 높은 활 성을 나타내는 것을 확인하였다. 도 2는 도1의 각 단계 분획물의 L-FABP 증가활성을 실험한 결과를 나타낸 도표이다.As a result of measuring the L-FABP increase activity according to the fraction, as shown in the first methanol extract (C-M), but showed a significant activity, it was confirmed that the highest activity in the final methanol fraction (M-M). Figure 2 is a chart showing the results of experiments to increase the L-FABP activity of each step fraction of FIG.
실시예 2 : 크로마토그래피를 이용한 L-FABP 증강물질의 정제Example 2 Purification of L-FABP Enhancer Using Chromatography
도 1과 같이 조제한 메탄올 분획물(M-M)로부터 다음과 같은 크로마토그래피에 의해 L-FABP 증강물질을 정제하였다. 우선 클로로포름으로 활성화된 실리카겔 60 컬럼에 시료인 메탄올 분획을 loading하고 클로로포름과 메탄올(CHCl3 : MeOH = 100 : 0 ∼ 0 : 100) 혼합용액으로 3 bed volume씩 전개한 후, L-FABP 증가활성을 검토하여 클로로포름과 메탄올 60 : 40에서 L-FABP 증가활성을 갖는 소분획(M-Ma)을 분리하고 이 분획물을 동일한 컬럼을 사용하여 클로로포름과 메탄올(CHCl3 : MeOH = 100 : 0 ∼ 0 : 100) 혼합용액으로 1 ∼ 5 bed volume씩 전개하면서 1 ∼ 2개의 분획으로 분리한 후 L-FABP 증가활성이 가장 높은 클로로포름과 메탄올 76 : 24 용출분획(M-Mb)을 분리하였다. 고활성의 상기 분획물을 ODS gel plate (20 ×10 cm), 메탄올 : 물 = 7 : 3의 전개용매와 에탄올에 녹인 5% H2SO4를 발색제로 사용하여 Prep-TLC를 실시하여 5개의 spot들(Rf value 순서에 따라 PG1 ~ PG5)을 분리하였다. 이중, 가장 높은 활성을 보인 분리된 소분획(PG2 :Rf value가 두 번째인 spot)을 HPLC(u-Bondapak C18, 3.9 ×150 mm)에 주입하고 물과 메탄올 gradient(H2O : MeOH = 100 : 0 ∼ 0 : 100) 조건에서 1 ml/min의 유속으로 용출하여, 활성분획(PG-2a)만을 농축한 후 활성분획을 동일한 조건에서 HPLC를 실시한 결과 좌우 대칭 성이 높은 단일 peak를 얻음으로써 비교적 정제도 높게 물질을 정제할 수 있었다. 도 3은 본 발명의 일 실시예로서 분획된 메탄올 추출물로부터 정제단계를 보인 과정도이고, 도 4는 도 3의 분리물질의 정제도를 보인 크로마토그램이다.L-FABP enhancer was purified from the methanol fraction (MM) prepared as shown in FIG. 1 by the following chromatography. First, the methanol fraction as a sample was loaded onto a silica gel 60 column activated with chloroform, and then developed in three bed volumes with a mixed solution of chloroform and methanol (CHCl 3 : MeOH = 100: 0∼0: 100), and then L-FABP increasing activity was observed. In this study, a small fraction (M-Ma) having L-FABP increasing activity was isolated from chloroform and methanol 60:40, and the fractions were separated using chloroform and methanol (CHCl 3 : MeOH = 100: 0-0: 100) using the same column. ) The mixture was separated into 1 to 2 fractions with 1 to 5 bed volumes, followed by separation of chloroform and methanol 76:24 elution fraction (M-Mb) with the highest L-FABP increasing activity. The highly active fractions were pre-tlced with ODS gel plate (20 × 10 cm), methanol: water = 7: 3 developing solvent, and 5% H 2 SO 4 dissolved in ethanol as a colorant. (PG1 to PG5) were separated according to the R f value order. Among them, the highest active fraction (PG2: spot with second R f value) was injected into HPLC (u-Bondapak C 18 , 3.9 × 150 mm), and water and methanol gradient (H 2 O: MeOH). = 100: 0 ~ 0: 100) eluted at a flow rate of 1 ml / min, concentrated only the active fraction (PG-2a), and HPLC was performed on the active fraction under the same conditions. By obtaining, the refinement | purification was comparatively high, and the substance was refined. Figure 3 is a process diagram showing the purification step from the methanol extract fractionated as an embodiment of the present invention, Figure 4 is a chromatogram showing the purification degree of the separation material of FIG.
실시예 3 : 도라지로부터 분리한 L-FABP 증강물질의 급성독성 실험Example 3 Acute Toxicity Test of L-FABP Enhancers Isolated from Bellflower
도라지에 함유된 L-FABP 증강물질의 급성독성을 조사하기 위해 랫트를 사용하여 경구투여한 결과 표1에 나타난 바와 같이 시료랑 0 ~ 3,000 mg/kg 농도의 모든 실험군에서 경구투여에 의한 독성은 나타나지 않았으며 따라서 LD50이 3,000 mg/kg 이상에 존재할 것으로 추정되었다(표1). To investigate the acute toxicity of L-FABP enhancer contained in the bellflower, oral administration of rats showed toxicity by oral administration in all experimental groups with 0 ~ 3,000 mg / kg concentration as shown in Table 1. Therefore, LD 50 was estimated to be above 3,000 mg / kg (Table 1).
실시예 4 : 도라지의 메탄올추출물 투여 쥐로부터 얻은 간기능개선 및 L-FABP 증강효과Example 4 Improvement of Liver Function and L-FABP Enhancement from Methanol Extract Administration Rats
실시예 1의 in vitro assay계에서 높은 L-FABP 증가활성을 보인 도라지의 in vivo 증가활성을 검토하기 위해 도라지 메탄올추출물을 쥐에게 0.15%와 0.3%의 농도로 5주간 액체식이로 공급한 후 혈액으로부터 VLDL-콜레스테롤, GOT 및 triglyceride를 측정하고, 간을 적출하여 다섯배의 potassium phosphate buffer(pH7.4, w/v)를 넣고 Teflon glass homogenizer(Glas-Col)로 균질화한 후, 10,000 rpm에서 원심분리하여 상등액을 취하고 다시 35,000 rpm에서 초원심분리하여 cytosol과 microsome을 분리하여 L-FABP와 CYP2E1 저해활성측정에 사용하였다. CYP2E1은 PNP(p-Nitrophenol 6-hydroxylation) 방법으로, L-FABP의 변화량은 실시예 1에서와 같은 ELISA 방법을 사용하여 측정하였다.In order to examine the in vivo increase activity of the bellflower showing high L-FABP increasing activity in the in vitro assay system of Example 1, the bellflower methanol extract was fed to rats in a liquid diet at 0.15% and 0.3% for 5 weeks, and then blood VLDL-cholesterol, GOT and triglyceride were measured from the liver, liver was extracted, added 5 times potassium phosphate buffer (pH7.4, w / v), homogenized with Teflon glass homogenizer (Glas-Col), and centrifuged at 10,000 rpm. The supernatant was isolated, ultracentrifuged at 35,000 rpm, cytosol and microsomes were isolated, and used for measuring L-FABP and CYP2E1 inhibitory activity. CYP2E1 was PNP (p-Nitrophenol 6-hydroxylation) method, and the amount of change in L-FABP was measured using the same ELISA method as in Example 1.
알코올에 의해 손상받은 간에서 발현이 증가되는 CYP2E1 활성은 p-nitrophenol(PNP) hydroxylation에 의하여 형성된 p-nitrocatechol을 측정하는 방법으로, 효소반응은 50 pmol 2E1, 5 mM PNP가 함유되어 있는 100 mM KP용액(pH 7.4)에 NADPH generating system(1 mM glucose 6-phosphate, 0.5 mM NADP+ and 1 U glucose-6-phosphatedehydogenase)의 첨가에 의하여 개시되며 100 ㎕ trichloroacetic acid(TCA, 20% v/v)에 의하여 중지시켰다. 5분간 10,000 ×g의 원심분리 후, 0.5 ml의 상등액을 얻고 2 M NaOH를 가하여 잘 섞은 후 각 시료를 535 nm에서 흡광도를 측정하였다.CYP2E1 activity, which is increased in alcohol-injured liver, is measured by p-nitrocatechol formed by p-nitrophenol (PNP) hydroxylation. The enzyme reaction is 100 mM KP containing 50 pmol 2E1 and 5 mM PNP. The solution was initiated by the addition of a NADPH generating system (1 mM glucose 6-phosphate, 0.5 mM NADP + and 1 U glucose-6-phosphatedehydogenase) in pH 7.4 and in 100 μl trichloroacetic acid (TCA, 20% v / v). Stopped. After centrifugation at 10,000 × g for 5 minutes, 0.5 ml of supernatant was obtained and mixed well by addition of 2 M NaOH, and then each sample was measured for absorbance at 535 nm.
그 결과 도라지 메탄올추출물은 0.3%의 투여농도에서 정상 대조군과 유사한 CYP2E1 발현억제활성을 나타냄으로써 알코올에 의한 간손상의 예방을 위한 기능성 신소재로 실용화될 가능성을 시사하였다(표2).As a result, the bellflower methanol extract showed a CYP2E1 expression inhibitory activity similar to that of the normal control group at a concentration of 0.3%, suggesting the possibility of practical use as a functional new material for preventing liver damage by alcohol (Table 2).
실시예 5: 도라지 추출물의 제제화 (정제)Example 5: Formulation of Bellflower Extract (Tablet)
하기의 배합의 정제를 통상의 타정기에 의해 제조하였다.Tablets of the following formulations were prepared by conventional tablet presses.
실시예 1의 도라지추출물의 분말 20질량부20 parts by mass of powder of bellflower extract of Example 1
덱스트린 72질량부Dextrin 72 parts by mass
분당(粉糖) 80질량부80 parts by mass per minute
글리세린지방산에스테르 8질량부8 parts by mass of glycerin fatty acid ester
원료의 혼합과 타정은 용이하고, 정미가 양호한 정제가 얻어졌다.Mixing and tableting of raw materials were easy, and refined refinement | purification was obtained.
실시예 6: 도라지 추출물의 제제화 (갭슐제)Example 6 Formulation of Bellflower Extract (Gap Capsule)
통상의 방법에 의해, 이하의 조성을 갖는 캡슐제를 제조하였다. 또한, 캡슐에는 1호 하드젤라틴캡슐을 사용하였다.By the usual method, the capsule which has the following compositions was manufactured. In addition, No. 1 hard gelatin capsule was used for the capsule.
<1캡슐(1정 200mg)중의 조성>Composition in 1 Capsule (200 mg)
실시예 1의 도라지추출물의 분말 5mg5 mg of bellflower extract of Example 1
콘스타치 60.0mgCornstarch 60.0mg
유당 100.0mgLactose 100.0mg
유산칼슘 10.0mgCalcium Lactate 10.0mg
하이드록시프로필셀룰로오스(HPC-L) 10.0mgHydroxypropyl cellulose (HPC-L) 10.0 mg
실시예 7: 도라지 추출물의 제제화 (과립)Example 7: Formulation of Bellflower Extract (Granules)
통상의 방법에 의해, 하기의 배합의 인스턴트티 과립을 유동층조립기에 의해 제조하였다.By a conventional method, instant tea granules of the following formulation were prepared by a fluid bed granulator.
실시예 1의 도라지추출물의 분말 20질량부20 parts by mass of powder of bellflower extract of Example 1
올리고당 40질량부40 parts by mass of oligosaccharide
구연산 50질량부50 parts by mass of citric acid
설탕 50질량부50 parts by mass of sugar
덱스트린 810질량부Dextrin 810 parts by mass
원료의 혼합과 유동층조립기에 의한 과립화는 용이하며, 정미가 양호한 인스턴트티과립이 얻어졌다.Mixing of the raw materials and granulation with a fluidized bed granulator were easy, and a fine grain of instant tea was obtained.
이상에서 살펴본 바와 같이, 본 발명 도라지로부터 분리한 간에서의 L-FABP 증강물질 및 그 정제방법은 blanching하여 분쇄한 후, 메탄올로 추출하여 그 잔사를 핵산, 클로로포름, 에틸아세테이트 순으로 분획하여 실리카겔 컬럼 크로마토그래피, 박층 클로마토그래피 및 HPLC 등 일련의 정제공정을 수행함으로써 얻어졌으며, 알코올로 인한 간의 지방축적이 감소됨으로써 지방간 예방용 기능성식품이나 약물에 사용될 수 있는 효과가 있게 되는 것이다. As described above, the L-FABP enhancer in liver isolated from the bellflower of the present invention and its purification method are blanched, pulverized, extracted with methanol, the residue is fractionated in the order of nucleic acid, chloroform, ethyl acetate, silica gel column It was obtained by performing a series of purification processes such as chromatography, thin layer chromatography, and HPLC, and by reducing the fat accumulation of liver due to alcohol, there is an effect that can be used in functional foods or drugs for preventing fatty liver.
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2004
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190046353A (en) * | 2017-10-26 | 2019-05-07 | 대한민국(농촌진흥청장) | The Black platycodon extract including alcoholic liver injury prevention functional ingredients, method for manufacturing thereof |
| KR102001525B1 (en) * | 2017-10-26 | 2019-07-19 | 대한민국 | The Black platycodon extract including alcoholic liver injury prevention functional ingredients, method for manufacturing thereof |
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| Publication number | Publication date |
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| KR20060015399A (en) | 2006-02-17 |
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