KR20060101049A - A method for producing antibiotic extracts from euonymus alatus and a seperation method for quercetin from the extracts - Google Patents
A method for producing antibiotic extracts from euonymus alatus and a seperation method for quercetin from the extracts Download PDFInfo
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- KR20060101049A KR20060101049A KR1020050022885A KR20050022885A KR20060101049A KR 20060101049 A KR20060101049 A KR 20060101049A KR 1020050022885 A KR1020050022885 A KR 1020050022885A KR 20050022885 A KR20050022885 A KR 20050022885A KR 20060101049 A KR20060101049 A KR 20060101049A
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- quercetin
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- 239000000284 extract Substances 0.000 title claims abstract description 109
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 title claims abstract description 68
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 34
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 title claims abstract description 34
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 229960001285 quercetin Drugs 0.000 title claims abstract description 34
- 235000005875 quercetin Nutrition 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title description 4
- 241000208368 Euonymus alatus Species 0.000 title description 2
- 230000003115 biocidal effect Effects 0.000 title description 2
- 239000000843 powder Substances 0.000 claims abstract description 93
- 230000000845 anti-microbial effect Effects 0.000 claims abstract description 42
- 241000880950 Viburnum dentatum Species 0.000 claims abstract description 16
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- 239000004599 antimicrobial Substances 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 35
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 14
- 238000004440 column chromatography Methods 0.000 claims description 10
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- 241000133122 Pluchea sericea Species 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 9
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- 239000003242 anti bacterial agent Substances 0.000 description 9
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- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
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- 239000011780 sodium chloride Substances 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- RMMXTBMQSGEXHJ-UHFFFAOYSA-N Aminophenazone Chemical compound O=C1C(N(C)C)=C(C)N(C)N1C1=CC=CC=C1 RMMXTBMQSGEXHJ-UHFFFAOYSA-N 0.000 description 3
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- 239000008272 agar Substances 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 2
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- 239000005711 Benzoic acid Substances 0.000 description 1
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 description 1
- 244000146486 Glehnia littoralis Species 0.000 description 1
- 235000004036 Glehnia littoralis Nutrition 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 208000034712 Rickettsia Infections Diseases 0.000 description 1
- 206010061495 Rickettsiosis Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
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- 238000010171 animal model Methods 0.000 description 1
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- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
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- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 229960003699 evans blue Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 229940124595 oriental medicine Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000026416 response to pain Effects 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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)
- A61K36/37—Celastraceae (Staff-tree or Bittersweet family), e.g. tripterygium or spindletree
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/21—Plant extracts
- A23V2250/2116—Flavonoids, isoflavones
- A23V2250/21168—Quercetin
-
- 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
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Alternative & Traditional Medicine (AREA)
- Mycology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
본 발명은 본 발명은 화살나무 유래의 항균 활성 추출분말을 제조하는 방법 및 상기 추출분말로부터 퀘르세틴을 분리하는 방법에 관한 것으로, 화살나무 각 부위별 추출분말을 제조하고 이로부터 퀘르세틴을 분리한 후 상기 추출분말이 항균 활성을 가질뿐만 아니라 특별히 상기 추출분말로부터 분리한 퀘르세틴은 획기적인 항균 활성을 가짐을 확인할 수 있으므로 항균 활성이 매우 뛰어난 제품을 제공하는 효과가 있다.The present invention relates to a method for preparing an antimicrobial active extract powder derived from arrowwood, and a method for separating quercetin from the extract powder. The extract powder for each part of the arrowwood is prepared and separated from the quercetin from the above. Not only has the extract powder has an antimicrobial activity, in particular, the quercetin isolated from the extract powder can be found to have a breakthrough antimicrobial activity, thus providing an excellent antimicrobial activity.
화살나무, 귀전우, 항균, 퀘르세틴 Arrowwood, Barr, Antibacterial, Quercetin
Description
본 발명은 화살나무로부터 항균 활성 추출분말을 제조하는 방법 및 상기 추출분말로부터 퀘르세틴을 분리하는 방법에 관한 것으로, 화살나무를 잎, 줄기 및 뿌리 부위별로 에탄올 추출한 후 에탄올을 증발시키고 증류수를 넣어 수용층을 얻어 이를 동결건조시킴으로써 항균 활성 추출분말을 제조하는 방법과 상기 각 부위별 추출분말을 분획한 후 크로마토그래피하여 퀘르세틴을 분리하는 방법에 관한 것이다.The present invention relates to a method for producing an antimicrobial active extract powder from arrowwood and a method for separating quercetin from the extract powder, after extracting ethanol by leaf, stem and root parts of arrowwood, ethanol is evaporated and distilled water is added to the aqueous layer. The present invention relates to a method for preparing an antimicrobial active extract powder by lyophilization and a method for separating quercetin by chromatography after fractionating the extract powder for each site.
화살나무(Euonymus alatus)는 노박 덩굴과에 속하는 낙엽관목으로서 높이가 3m에 달하며 한국, 일본, 사할린, 중국 지역에 분포한다. 식물 전체는 반들반들하고 털이 없으며 많이 분지되어 있다. 작은 가지는 네모나고 보통 녹색을 띤다. 튼튼한 가지에는 보통 납작한 나뭇가지 모양의 코르크질 날개가 붙어 있는데 날개의 너비는 1cm에 달하고 갈색이다. 참빗나무, 홋잎나무라고도 부린다. 잎은 마디마디 2장이 마주 붙으며 잎 모양은 달걀 꼴로 양끝이 뾰족하다. 잎의 길이는 3~5cm이고 가장자리에 작은 톱니가 있으며 잎 뒷면은 잿빛을 띤 녹색이다. 어린잎은 나물로 한다. 잎에는 에피프리에델라놀, 프리에델린, 퀘르세틴, 둘시톨(dulcitol) 등이 함유되어 있다. 5월에 연한 녹색의 꽃이 피어 가을에 둥글납작한 열매가 갈색으로 익는다. 종자유에는 포화지방산, 올레인산, 리놀산, 리놀렌산, 카프릭산 및 안식향산 등이 함유되어 있다. 줄기에 붙어 있는 날개의 생김새가 특이하여 귀전우, 신전목이라고 부른다. 한방에서는 지혈, 구어혈, 통경에 사용한다. Euonymus alatus is a deciduous shrub belonging to Novak vine family, 3m high and distributed in Korea, Japan, Sakhalin and China. The whole plant is silky, hairless and much branched. Small branches are square and usually green. Sturdy branches usually have flat twig cork wings that are 1 cm wide and brown. It is also called a true tree or a leaf tree. The leaf has two nodes facing each other, and the leaf shape is egg-shaped and pointed at both ends. The leaves are 3 ~ 5cm long, with small teeth on the edges, and the back of the leaves is ash green. Young leaves are made of herbs. The leaves contain epipriedellanol, priedelin, quercetin, and dulcitol. Light green flowers bloom in May and round fruits ripen brown in autumn. Seed oil contains saturated fatty acids, oleic acid, linoleic acid, linolenic acid, capric acid and benzoic acid. The appearance of the wing attached to the stem is unusual, so it is called a deciduous cow, a shrine. Oriental medicine is used for hemostasis, gore blood, pain.
항생제는 미생물에 의해 만들어져 미생물 및 그 밖의 세포의 발육·기능을 저해하는 물질로서 동·식물에서 추출되는 항균물질을 포함시키기도 한다. 1941년 미국의 S.A. 왁스먼은 세균에 의해 생합성되는 물질로서 다른 미생물의 생장을 저해하는 물질을 ＜antibiotic＞으로 정의하였으나 그 뒤 항종양·항바이러스 항생물질이 발견되어 왁스먼이 제창한 항생물질의 정의는 항균성항생물질로 한정되고, 현재와 같은 개념으로 확정되었다. 또 미생물이 만드는 물질 속에는 항균작용을 나타내지 않고 효소저해작용과 특이적 약리작용을 가진 물질도 발견되어 의약품으로서의 효능이 주목되고 있다. 이 물질들은 생산하는 미생물의 생육에 필수적인 것이 아니라는 의미에서 ＜미생물의 2차대사산물＞이라고 불리며, 항생물질도 이 가운데 포함된다. 지금까지 발견된 항생물질수는 4000을 넘으며 3만 이상의 유도체가 만들어졌고 50종 이상이 임상적으로 사용되고 있다. 항생물질의 발견으로 인류는 비로소 세균·리케차감염증을 극복하고, 곰팡이·바이러스성질환의 치료도 기대하게 되었다. 오늘날 항생물질은 사람·가축의 의약품뿐만 아니라 농약이나 발육촉진을 목적으로 한 가축사료첨가제 등으로 널리 사용된다. Antibiotics are substances produced by microorganisms that inhibit the development and function of microorganisms and other cells, and may include antimicrobial substances extracted from animals and plants. In 1941, S.A. Waxman defined a substance that is biosynthesized by bacteria as an antibiotic substance. However, since anti-tumor and antiviral antibiotics were discovered, the definition of antibiotics suggested by Waxman was antimicrobial antibiotics. It was limited to and was confirmed with the present concept. In addition, the substance produced by the microorganism does not show antimicrobial action, but also has a substance with enzymatic inhibition and specific pharmacological action has attracted attention as a drug. These substances are called <secondary metabolites of microorganisms> in that they are not essential for the growth of the microorganisms they produce, and antibiotics are included among them. The number of antibiotics found so far is over 4000, with over 30,000 derivatives made and over 50 clinically used. The discovery of antibiotics has led humans to overcome bacterial and Rickettsia infections and to anticipate the treatment of fungal and viral diseases. Today, antibiotics are widely used not only for human and livestock medicines, but also for pesticides and animal feed additives for the purpose of promoting development.
본 발명자는 상기와 같은 점을 고려하여 화살나무로부터 항균 활성이 우수한 퀘르세틴(quercetin)을 효과적으로 분리 및 대량생산해내는 방법을 연구한 결과, 화살나무의 잎, 줄기 또는 뿌리를 부위별로 분리하여 에탄올 추출한 후 에탄올을 완전 증발시켜 각 부위별 추출물을 얻은 후 상기 각 부위별 추출물에 증류수를 첨가하여 혼합한 후 층분리를 시킨 다음 수용층을 분리하여 이를 동결건조하여 각 부위별 추출분말을 얻은 다음 상기 각 부위별 추출분말에 물를 첨가하여 혼합한 다음 이를 컬럼 크로마토그래피용 비이온성 수지로 흡착시킨 후 용매를 클로로포름, 클로로포름: 에틸 아세테이트 1:4 혼합용액, 에틸 아세테이트, 메탄올의 순서로 사용하여 용출시킴으로써 각 부위의 용매별 분취액들을 얻고 상기 각 부위의 용매별 분취액들에 대하여 항균활성을 조사하여 항균활성을 보이는 각 부위의 메탄올 분획을 분리한 뒤 상기 각 부위의 메탄올 분획을 다시 세파덱스 엘에이치-20 컬럼 크로마토그래피를 수행하여 항균 활성이 우수한 퀘르세틴을 분리할 수 있음을 발견하고 본 발명을 완성하였다.In view of the above, the present inventors studied a method of effectively separating and mass-producing quercetin having excellent antimicrobial activity from arrowheads. Ethanol was completely evaporated to obtain an extract for each site, and then distilled water was added and mixed with the extract for each site, followed by layer separation. The aqueous layer was separated and lyophilized to obtain an extract powder for each site. Water was added to the extract powder and mixed, followed by adsorption with a nonionic resin for column chromatography. The solvent was eluted by using a solvent of chloroform, chloroform: ethyl acetate 1: 4 mixed solution, ethyl acetate and methanol in this order. Obtain aliquots of the aliquots and relate to the aliquots of the solvents at each site. After investigating the activity, the methanol fraction of each site showing antimicrobial activity was isolated, and then the methanol fraction of each site was separated and Separdex LS-20 column chromatography was found to be able to separate quercetin having excellent antibacterial activity. The invention has been completed.
따라서, 본 발명의 목적은 화살나무로부터 항균 활성 추출분말을 제조하는 방법을 제공하는데 있다.Accordingly, it is an object of the present invention to provide a method for producing an antimicrobial active powder from arrowwood.
본 발명의 다른 목적은 화살나무로부터 얻은 항균 활성 추출분말로부터 천연 항균제로서의 퀘르세틴을 분리하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for separating quercetin as a natural antimicrobial agent from an antimicrobial active extract powder obtained from arrowwood.
본 발명의 상기 목적은 화살나무를 이용하여 추출분말을 제조하고 이로부터 퀘르세틴을 분리한 다음 디스크 확산법 및 액체배지 희석법을 이용하여 상기 화살나무 유래 추출분말 및 퀘르세틴의 항균성을 조사하고 모세혈관 투과 억제도 측정법을 통해 항염증 활성을 조사함으로써 달성하였다.The object of the present invention is to prepare an extract powder using arrowwood and to separate the quercetin from it and to investigate the antimicrobial activity of the arrowwood-derived extract powder and quercetin using a disk diffusion method and liquid medium dilution method and to inhibit capillary permeation Achieved by investigating anti-inflammatory activity via assay.
이하 본 발명의 구성을 설명한다.Hereinafter, the configuration of the present invention.
본 발명은 화살나무를 이용하여 추출분말을 제조하고 이로부터 퀘르세틴을 분리하는 단계; 디스크 확산법을 이용하여 화살나무 유래 추출분말 및 퀘르세틴의 항균성을 조사하는 단계; 액체배지 희석법을 이용하여 화살나무 유래 추출분말 및 퀘르세틴의 항균성을 조사하는 단계; 및 모세혈관 투과 억제도 측정법을 통해 화살나무 유래 추출분말 및 퀘르세틴의 항염증 활성을 조사하는 단계로 구성된다.The present invention comprises the steps of preparing an extract powder using an arrow tree and separating the quercetin from it; Investigating the antimicrobial activity of quercetin-derived extract powder and quercetin using a disk diffusion method; Investigating the antimicrobial activity of quercetin-derived extract powder and quercetin using a liquid medium dilution method; And examining the anti-inflammatory activity of quercetin-derived extract powder and quercetin through capillary permeability inhibition assay.
본 발명은 화살나무의 잎, 줄기 또는 뿌리를 부위별로 분리하여 에탄올 추출 한 후 에탄올을 완전 증발시켜 각 부위별 추출물을 얻는 단계; 및The present invention comprises the steps of separating the leaves, stems or roots of the arrow tree by the ethanol extract and then ethanol completely evaporated to obtain an extract for each site; And
상기 각 부위별 추출물에 증류수를 첨가하여 혼합한 후 층분리를 시킨 다음 수용층을 분리하여 이를 동결건조하여 각 부위별 추출분말을 얻는 단계를 포함하는 화살나무 유래 항균 활성 추출분말 제조방법을 제공한다.Distilled water is added to the extract for each site, the mixture is separated, the layer is separated, and then the aqueous layer is separated and lyophilized to provide an extract from the arrowwood-derived antimicrobial active powder comprising the step of obtaining an extract powder for each site.
본 발명은 또한 화살나무의 잎, 줄기 또는 뿌리를 부위별로 분리하여 에탄올 추출한 후 에탄올을 완전 증발시켜 각 부위별 추출물을 얻는 단계;The present invention also comprises the steps of separating the leaves, stems or roots of the arrow tree by ethanol extraction and then completely evaporated ethanol to obtain an extract for each site;
상기 각 부위별 추출물에 증류수를 첨가하여 혼합한 후 층분리를 시킨 다음 수용층을 분리하여 이를 동결건조하여 각 부위별 추출분말을 얻는 단계;Adding distilled water to the extract for each site, mixing the layers, separating the aqueous layer, and lyophilizing it to obtain an extract powder for each site;
상기 각 부위별 추출분말에 물을 첨가하여 혼합한 다음 이를 컬럼 크로마토그래피용 비이온성 수지로 흡착시킨 후 용매를 클로로포름, 클로로포름: 에틸 아세테이트 1:4 혼합용액, 에틸 아세테이트, 메탄올의 순서로 사용하여 용출시킴으로써 각 용매별 분취액들을 얻는 단계;Water was added to each of the extracted powders for mixing, followed by adsorption with a nonionic resin for column chromatography, and the solvent was eluted using chloroform, chloroform: a mixture of ethyl acetate 1: 4 solution, ethyl acetate, and methanol. To obtain aliquots for each solvent;
상기 각 용매별 분취액들에 대하여 항균활성을 조사하여 항균활성을 보이는 메탄올 분획을 분리하는 단계; 및Irradiating antimicrobial activity on the aliquots of each solvent to separate the methanol fraction showing antimicrobial activity; And
상기 메탄올 분획을 다시 세파덱스 엘에이치-20 컬럼 크로마토그래피를 수행하여 항균 활성을 보이는 분획을 모으는 단계를 포함하는 화살나무 유래 항균 활성 퀘르세틴의 분리방법을 제공한다.The methanol fraction is subjected to Sephadex L-20 column chromatography again to provide a method of separating the antibacterial activity quercetin derived from arrowwood comprising the step of collecting the fraction showing the antimicrobial activity.
이하, 본 발명에 대해 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 화살나무의 잎, 줄기 또는 뿌리를 부위별로 분리하여 수세한 후 세절한 다음 건조시킨 뒤 건조된 화살나무 각 부위를 에탄올에 넣어 50-70℃에서 6-12시간 추출한 후 거즈로 여과한 다음 에탄올을 완전 증발시켜 각 부위별 추출물을 얻는 단계; 및In the present invention, the leaves, stems or roots of the arrow tree are separated by water, washed with water, and then dried, and then dried in each part of the dried arrow tree in ethanol for 6-12 hours, and filtered with gauze. Then completely evaporating ethanol to obtain an extract for each site; And
상기 각 부위별 추출물에 증류수를 첨가하여 혼합한 후 층분리를 시킨 다음 수용층을 분리하여 이를 동결건조하여 각 부위별 추출분말을 얻는 단계를 포함하는 화살나무 유래 항균 활성 추출분말 제조방법에 관한 것이다.It relates to a method for producing an antimicrobial active extract powder derived from arrowwood comprising the step of adding distilled water to the extract for each site and then mixing the layers and then separating the aqueous layer and lyophilizing it to obtain an extract powder for each site.
더 나아가, 본 발명은 상기 각 부위별 추출분말을 물에 첨가하여 혼합한 다음 이를 컬럼 크로마토그래피용 비이온성 수지로 흡착시킨 후 용매를 클로로포름, 클로로포름: 에틸 아세테이트 1:4 혼합용액, 에틸 아세테이트, 메탄올의 순서로 사용하여 용출시킴으로써 각 부위의 각 용매별 분취액들을 얻는 단계;Furthermore, in the present invention, the extract powder for each site is added to water, mixed, and then adsorbed with a nonionic resin for column chromatography, and then the solvent is chloroform, chloroform: ethyl acetate 1: 4 mixed solution, ethyl acetate, methanol Obtaining an aliquot of each solvent of each site by eluting using in the order of;
상기 각 부위의 각 용매별 분취액들에 대하여 디스크 확산법을 통하여 항균활성을 조사하여 가장 높은 항균활성을 보이는 메탄올 분획을 분리하는 단계; 및Investigating the antimicrobial activity of the aliquots of each solvent by the disk diffusion method to separate the methanol fraction showing the highest antimicrobial activity; And
상기 메탄올 분획을 다시 세파덱스 엘에이치-20 컬럼 크로마토그래피를 수행하여 각 부위별로 각각 5개씩의 분획들을 얻고 이들 분획들 중 디스크 확산법을 통하여 각 부위별로 가장 좋은 항균 활성을 보이는 분획들을 모으는 단계를 포함하는 화살나무 유래 항균 활성 퀘르세틴의 분리방법에 관한 것이다.Separating the methanol fractions again, Sepadex LS-20 column chromatography was performed to obtain five fractions for each site, and the fractions having the best antimicrobial activity for each site were collected through the disk diffusion method. The present invention relates to a method for isolating antibacterial quercetin derived from arrowwood.
본 발명에서 퀘르세틴의 분리시 항균 활성을 조사하기 위한 방법으로는 당업계에 알려진 다양한 방법들을 모두 이용할 수 있으며 본 발명에서는 간단한 디스크 확산법을 사용하였다.As a method for investigating the antibacterial activity of the quercetin in the present invention, various methods known in the art may be used, and in the present invention, a simple disk diffusion method was used.
이하 본 발명을 다음과 같은 실시예 및 실험예에 의하여 더욱 상세하게 설명하고자 한다. 단, 다음의 실시예 및 실험예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이 것들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only for illustrating the present invention, the scope of the present invention is not limited to these.
실시예 1: 화살나무 추출분말 제조 및 이로부터 퀘르세틴의 분리Example 1 Preparation of Arrowwood Extract Powder and Separation of Quercetin from It
화살나무 잎, 줄기 및 뿌리 부위별로 분리하여 수세한 후 세절한 다음 더 이상 중량의 변화가 없을 때까지 그늘에서 건조시킨 뒤 건조된 화살나무 각 부위 1kg씩을 에탄올 1L에 각각 넣어 60℃에서 8시간 추출한 후 거즈로 여과 후, 감압농축기(Rotary Vacuum Evaporator; Heidolph WB 2000, Germany)를 이용하여 에탄올을 완전 증발시켜 각 부위별 추출물을 얻었다.After washing by dividing by arrow leaf, stem and root area, washing it, and then drying it in the shade until there is no change in weight, 1kg each dried arrowwood part is put in 1L of ethanol and extracted for 8 hours at 60 ℃. After filtration with gauze, ethanol was evaporated completely using a rotary vacuum evaporator (Heidolph WB 2000, Germany) to obtain an extract for each site.
상기 각 부위별 추출물에 1L 증류수를 첨가하여 혼합한 후 층분리를 시킨 다음 수용층을 분리하여 이를 동결건조하여 각 부위별 추출분말을 제조하였다.1L distilled water was added to each part of the extract for mixing, followed by layer separation, and then the aqueous layer was separated and lyophilized to prepare an extract powder for each part.
상기 각 부위별 추출분말에 물 3L를 첨가하여 혼합한 다음 이를 컬럼 크로마토그래피용 비이온성 수지로 흡착시킨 후 용매를 클로로포름, 클로로포름: 에틸 아세테이트 1:4 혼합용액, 에틸 아세테이트, 메탄올의 순서로 사용하여 용출시켰다. 각 부위의 용매별 분취액들에 대하여 디스크 확산법을 실시하여 가장 우수한 항균활성을 보이는 각 부위의 메탄올 분획을 다시 세파덱스 엘에이치-20 컬럼 크로마토 그래피를 수행하여 각 부위별로 5개씩의 분획들을 얻었다. 상기 분획들에 대하여 다시 디스크 확산법을 실시하여 우수한 항균활성을 보이는 각 부위별 분획들을 모았다.After adding 3 L of water to each part of the extraction powder and mixing the mixture, it was adsorbed with a nonionic resin for column chromatography, and then the solvent was used in the order of chloroform, chloroform: ethyl acetate 1: 4 mixed solution, ethyl acetate, and methanol. Eluted. Solvent aliquots of each site were subjected to disk diffusion and the methanol fraction of each site showing the best antimicrobial activity was again subjected to Sephadex L-20 column chromatography to obtain 5 fractions of each site. Disc diffusion was performed again on the fractions to collect fractions for each site showing excellent antimicrobial activity.
디스크 확산법(Disc diffusion method)은 감수성 검사용 배지로는 무엘러 힌톤 아가(Mueller Hinton agar)를 사용하고 시험 균주로서 칸디다 알비칸스(Candida albicans) KCTC 1040을 사용하여 상기 배지에 상기 시험 균주를 전배양한 후 이를 본배양배지에 접종하여 균주를 배양하면서 균주의 성장기 중 대수증식기 상태에 도달한 균을 이용하여 항균활성을 측정하였다. 배양된 균주를 페트리상의 고체배지에 접종하여 골고루 퍼지게 한 후 추출물을 필터 페이퍼 디스크(filter paper disc)에 흡수시켜 고체배지 표면 위에 놓아 48시간동안 혐기성 챔버(anaerobic chamber)에서 배양한 후 항균력을 디스크 주위 클리어 존(clear zone)의 유무 및 크기로써 확인하였다.Disc diffusion method is pre-cultivation of the test strain in the medium using Mueller Hinton agar as a susceptibility test medium and Candida albicans KCTC 1040 as a test strain After inoculating them into the culture medium, the strains were cultured and the antimicrobial activity was measured using the bacteria that reached the logarithmic growth state of the strain. The cultured strain was inoculated in a solid medium on Petri to spread it evenly, and then the extract was absorbed in a filter paper disc, placed on the surface of the solid medium, incubated in an anaerobic chamber for 48 hours, and then the antibacterial activity was observed around the disk. It was confirmed by the presence and size of the clear zone (clear zone).
상기에서 얻은 분획의 물리화학적 및 분광학적 특징을 조사한 결과 하기 화학식의 퀘르세틴(quercetin)임을 확인하였다.Examination of the physicochemical and spectroscopic characteristics of the obtained fractions confirmed that it was quercetin of the following formula.
실험예 1: 디스크 확산법을 이용한 화살나무 유래 추출분말 및 퀘르세틴의 항균성 조사Experimental Example 1: Investigation of antimicrobial activity of quercetin-derived extract powder and quercetin using disk diffusion method
디스크 확산법(Disc diffusion method)에 의한 감수성 검사용 배지로는 무엘러 힌톤 아가(Mueller Hinton agar)를 사용하였다. 시험 균주를 전배양한 후 이를 본배양배지에 접종하여 균주를 배양하면서 균주의 성장기 중 대수증식기 상태에 도달한 균을 이용하여 항균활성을 측정하였다. 배양된 균주를 페트리상의 고체배지에 접종하여 골고루 퍼지게 한 후 추출물을 필터 페이퍼 디스크(filter paper disc)에 흡수시켜 고체배지 표면 위에 놓아 48시간동안 혐기성 챔버(anaerobic chamber)에서 배양한 후 항균력을 디스크 주위 클리어 존(clear zone)의 유무로써 확인하였다.Mueller Hinton agar was used as a medium for susceptibility testing by the disc diffusion method. After the pre-cultivation of the test strain was inoculated into the main culture medium and cultured the strain, the antibacterial activity was measured by using the bacteria that reached the logarithmic growth state in the growth phase of the strain. The cultured strain was inoculated in a solid medium on Petri to spread it evenly, and then the extract was absorbed in a filter paper disc, placed on the surface of the solid medium, incubated in an anaerobic chamber for 48 hours, and then the antibacterial activity was observed around the disk. It was confirmed by the presence or absence of a clear zone.
추출물의 항균효과를 검토하기 위한 시험균주로는 그람양성세균으로 스타피로코커스 아우레우스(Staphylococcus aureus) KCTC 1621, 음성세균으로 살모넬라 티피무리움(Salmonella typhimurium) KCTC 2057은 및 효모류로 칸디다 알비칸스(Candida albicans) KCTC 1040을 사용하였다.As a test strain for examining the antimicrobial effect of the extract, Gram-positive bacteria were Staphylococcus aureus KCTC 1621, Salmonella typhimurium KCTC 2057 as a negative bacteria and Candida albicans (Yeast) Candida albicans ) KCTC 1040 was used.
실험종료 후 측정한 결과를 하기 표 1 내지 표 3에 나타내었다.The results measured after the end of the experiment are shown in Tables 1 to 3 below.
상기 표 1 내지 표 3을 통해 알 수 있듯이, 칸디디아시스(candidiasis)의 일반적인 병원체인 칸디다 알비칸스 KCTC 1040(Candida albicans KCTC 1040)은 퀘르세틴 투여군에서 가장 항균활성을 나타내었다. 식중독 원인 병원성 장내세균인 살모넬라 티피무리움(Salmonella typhimurium) KCTC 2057 및 그람양성의 화농성질환 병원균이며, 식중독 원인균인 스타피로코커스 아우레우스(Staphylococcus aureus) KCTC 1621에서도 화살나무 유래 추출분말에서 비교적 높은 항균활성을 나타내었으며 특히 퀘르세틴 투여군에서 월등히 우수한 항균활성이 나타남을 알 수 있었다.As can be seen from Table 1 to Table 3, Candida albicans KCTC 1040 ( Candida albicans KCTC 1040), a common pathogen of candidiasis showed the most antibacterial activity in the quercetin-administered group. Salmonella typhimurium KCTC 2057, a pathogenic intestinal bacterium causing food poisoning, and a Gram-positive purulent disease pathogen, and Staphylococcus aureus KCTC 1621, a bacterium that causes food poisoning, are relatively high In particular, the quercetin-administered group showed excellent antimicrobial activity.
실험예 2: 액체배지 희석법(Broth dilution method)을 이용한 항균성 조사Experimental Example 2: Investigation of antimicrobial activity by using broth dilution method
각 부위별 추출분말과 퀘르세틴 분획을 농도별로 함유한 무엘러 힌톤 브로쓰(Mueller Hinton broth)에 시험균 1%를 넣어 18시간동안 각 시험균의 적정 배양온도인 혐기성 챔버(anaerobic chamber)에서 배양한 후 균의 증식 유무를 관찰하여 세균이 증식하지 않은 추출물의 최소량을 최저발육억제농도(MIC)로 정하여 항균성을 조사하였다. 1% of the test bacteria was added to Mueller Hinton broth containing extract powder and quercetin fraction for each site, followed by incubation in an anaerobic chamber for 18 hours. After observing the growth of bacteria, the antimicrobial activity was investigated by setting the minimum amount of the growth inhibitory concentration (MIC) as the minimum amount of the extract which did not grow.
시험균주로는 그람양성세균으로 스타피로코커스 아우레우스(Staphylococcus aureus) KCTC 1621, 음성세균으로 살모넬라 티피무리움(Salmonella typhimurium) KCTC 2057은 및 효모류로 칸디다 알비칸스(Candida albicans) KCTC 1040을 사용하였다. Test strains were Staphylococcus aureus KCTC 1621 as Gram-positive bacteria, Salmonella typhimurium KCTC 2057 as negative bacteria, and Candida albicans KCTC 1040 as yeast. .
실험종료 후 측정한 결과를 하기 표 4 내지 표 6에 나타내었다.The results measured after the end of the experiment are shown in Tables 4 to 6 below.
실험예 3: 모세혈관 투과 억제도 측정법을 통한 항염증 활성 조사Experimental Example 3: Investigation of anti-inflammatory activity through capillary permeability inhibition assay
상기 실시예 1에서 제조된 화살나무의 각 부위별 추출분말과 퀘르세틴 분획의 항염증 활성을 조사하였다. 항염증 활성 측정은 염증반응 1기에 염증부위로의 모세혈관 투과도가 증가하는 현상을 저해하는 정도를 측정하는 모세혈관 투과 억제도 측정법(Whittle 등)으로 측정하였다.The anti-inflammatory activity of the extract powder and quercetin fraction of each part of the arrowwood prepared in Example 1 was investigated. Anti-inflammatory activity was measured by capillary permeability inhibition assay (Whittle et al.) To measure the degree of inhibition of the increase in capillary permeability to the inflammatory site in the first stage of the inflammatory response.
실험동물은 5주령된 수컷 ICR계 마우스를 사용하였고 동물 사육조건은 온도 24 ± 2℃, 상대 습도 57 ± 5 %, 명암교대 12시간으로 하였다. 물과 사료는 자유롭게 섭취할 수 있도록 충분히 공급하여 일주인간 사육하였다.5 weeks old male ICR mice were used for the experimental animals. Animal breeding conditions were 24 ± 2 ° C, 57 ± 5% relative humidity, and 12 hours of contrast. Water and feed were fed enough to be freely ingested for one week.
상기와 같이 예비 사육된 ICR계 마우스에 식염수, 각 부위별 추출분말, 각 부위별 추출분말의 혼합물, 퀘르세틴 분획 및 양성대조약인 아미노피린(aminopyrine)을 경구 투여한 다음 1시간 후 생리식염수에 녹여 제조한 1% 에반스 블루(evans blue)를 마우스 몸무게 10g당 0.1 mL의 용량으로 꼬리 정맥내에 정맥주사하고 25분 후 생리 식염수에 희석하여 제조한 1 % 아세트산(acetic acid)을 마우스 몸무게 10g당 0.1 mL의 용량으로 복강내 주사하였다. 10분 후 염증 유발시 수반되는 통증에 대한 반응을 꼬임수(writhing number)로 10분 동안 측정하였고 측정 후 즉시 경추 탈골법으로 실험동물을 도태시킨 후 복강내로 투과된 에반스 블루(evans blue)를 취하기 위해 5 mL의 생리식염수를 복강에 가하고 복부를 가볍게 흔들어준 후 세척액을 취하였다. 3000 rpm으로 15분 동안 원심분리한 후 상층액을 취하여 분광광도계로 590 nm에서 흡광도를 측정하여 대조군과 비교하였다.Prepared by dissolution in saline, saline, extract powder for each site, mixture of extract powder for each site, quercetin fraction and aminopyrine (positive control) aminopyrine (1 hour) after 1 hour 0.1 mL of 1% acetic acid prepared by intravenous intravenous injection of tail vein at a dose of 0.1 mL per 10 g of mouse weight and diluted 25 minutes later with physiological saline. Intraperitoneal injection at dose. After 10 minutes, the response to pain associated with inflammation was measured for 10 minutes by writhing number, and immediately after the measurement, the animal was selected by cervical dislocation and the evans blue permeated into the abdominal cavity. For 5 mL of saline solution was added to the abdominal cavity, gently shake the abdomen, and the washing solution was taken. After centrifugation at 3000 rpm for 15 minutes, the supernatant was taken and measured for absorbance at 590 nm with a spectrophotometer to compare with the control.
실험 결과, 표 7에 나타낸 바와 같이 각각의 추출분말 및 혼합물 그리고 분획에서 대조군인 식염수보다 유의적으로 모세관 투과 억제도가 높게 나타나고 통증 횟수가 적게 나타났으며 특별히 줄기와 뿌리 추출분말의 혼합물과 퀘르세틴 투여군에서 월등히 높은 모세관 투과 억제도를 나타내고 통증 횟수도 현저히 감소되었음을 알 수 있었다.As a result, as shown in Table 7, each extract powder, mixture and fraction showed significantly higher capillary permeability inhibition and fewer pains than the saline control group, especially the mixture of stem and root extract powder and quercetin-treated group. Showed a significantly higher degree of suppression of capillary permeation and a significant decrease in the number of pains.
이상 상기에서 살펴본 바와 같이 본 발명은 화살나무 유래의 항균 활성 추출분말을 제조하는 방법 및 상기 추출분말로부터 퀘르세틴을 분리하는 방법에 관한 것으로, 화살나무 각 부위별 추출분말을 제조하고 이로부터 퀘르세틴을 분리한 후 상기 추출분말이 항균 활성을 가질뿐만 아니라 특별히 상기 추출분말로부터 분리한 퀘르세틴은 획기적인 항균 활성을 가짐을 확인할 수 있으므로 식품 및 의약산업상 매우 유용한 것이다.As described above, the present invention relates to a method for preparing an antimicrobial active extract powder derived from arrowwood and a method for isolating quercetin from the extract powder. After that, the extract powder not only has antimicrobial activity but also specifically, the quercetin isolated from the extract powder can be found to have breakthrough antimicrobial activity, which is very useful in the food and pharmaceutical industries.
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| KR102078414B1 (en) * | 2018-10-31 | 2020-02-17 | 주식회사 발효예스 | METHOD FOR MANUFACTURING Euonymus alatus FERMENTED BROTH, AND THE Euonymus alatus FERMENTED BROTH MANUFACTURED BY THE METHOD |
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| WO2015105373A1 (en) * | 2014-01-09 | 2015-07-16 | 한국생명공학연구원 | Composition for prevention or treatment of asthma, comprising e uonymus alatus extract or fraction thereof |
| KR102078414B1 (en) * | 2018-10-31 | 2020-02-17 | 주식회사 발효예스 | METHOD FOR MANUFACTURING Euonymus alatus FERMENTED BROTH, AND THE Euonymus alatus FERMENTED BROTH MANUFACTURED BY THE METHOD |
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