KR20150135884A - Preparation method for tea Catechin haaving high pure Epigallocatechin gallate - Google Patents
Preparation method for tea Catechin haaving high pure Epigallocatechin gallate Download PDFInfo
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- 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 title claims abstract description 48
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229940030275 epigallocatechin gallate Drugs 0.000 title abstract description 36
- 238000002360 preparation method Methods 0.000 title description 2
- 244000269722 Thea sinensis Species 0.000 claims abstract description 24
- 235000009569 green tea Nutrition 0.000 claims abstract description 16
- 239000000284 extract Substances 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 229960001948 caffeine Drugs 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 12
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims description 11
- 235000005487 catechin Nutrition 0.000 claims description 11
- 150000001765 catechin Chemical class 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 8
- 235000013616 tea Nutrition 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000004440 column chromatography Methods 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 6
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 4
- 239000012074 organic phase Substances 0.000 claims description 4
- 238000003287 bathing Methods 0.000 claims description 3
- 239000002131 composite material Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 30
- 238000010791 quenching Methods 0.000 description 9
- 230000000171 quenching effect Effects 0.000 description 9
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 description 8
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 5
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 description 4
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 description 4
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 4
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 4
- XMOCLSLCDHWDHP-UHFFFAOYSA-N L-Epigallocatechin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-UHFFFAOYSA-N 0.000 description 4
- LPTRNLNOHUVQMS-UHFFFAOYSA-N epicatechin Natural products Cc1cc(O)cc2OC(C(O)Cc12)c1ccc(O)c(O)c1 LPTRNLNOHUVQMS-UHFFFAOYSA-N 0.000 description 4
- 235000012734 epicatechin Nutrition 0.000 description 4
- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 3
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 229950001002 cianidanol Drugs 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- OEIJRRGCTVHYTH-UHFFFAOYSA-N Favan-3-ol Chemical compound OC1CC2=CC=CC=C2OC1C1=CC=CC=C1 OEIJRRGCTVHYTH-UHFFFAOYSA-N 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930182497 flavan-3-ol Natural products 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
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)
- A61K36/82—Theaceae (Tea family), e.g. camellia
-
- 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
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
-
- 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
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
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- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
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Abstract
본 발명은 녹차잎으로부터 하기의 구조식의 EGCG(Epigallocatechin gallate)를 고순도로 함유하는 차카테킨 혼합추출물의 제조방법에 관한 것이다.The present invention relates to a method for producing a tea catechin mixture extract containing high purity Epigallocatechin gallate (EGCG) of the following formula from green tea leaves.
Description
본 발명은 녹차잎으로부터 하기의 구조식의 EGCG(Epigallocatechin gallate)를 고순도로 함유하는 차카테킨 혼합추출물의 제조방법에 관한 것이다.The present invention relates to a method for producing a tea catechin mixture extract containing high purity Epigallocatechin gallate (EGCG) of the following formula from green tea leaves.
녹차는 국내외에서 널리 사용되고 있는 식품 소재로서, 원료의 주요 공급지로 중국이 잘 알려져 있으며 일본과 미국에서 큰 시장을 형성하고 있다. 최근에는 녹차의 물 또는 주정 추출물이 식품 원료로 사용되고 있다. 뿐만 아니라 차에서 추출한 카테킨은 “차카테킨”으로 식품첨가물로 허용되어 있는데, 이는 폴리페놀 화합물의 일종으로 특유의 쓴맛을 가지고 있으며 flavan-3-ol을 기본구조로 하고 있다.Green tea is widely used in domestic and overseas food materials, China is well known as a major source of raw materials, and has formed a large market in Japan and the United States. In recent years, green tea or water extracts have been used as food ingredients. In addition, catechins extracted from tea are accepted as food additives as "tea catechins", a type of polyphenolic compound with a characteristic bitter taste and a basic structure of flavan-3-ol.
녹차의 인체에 대한 건강 효능은 세계적으로 주목을 받고 있고, 세계 각국에서 생체 효능에 대한 연구가 활발히 진행되고 있다. 녹차 카테킨 중 EGCG(Epigallocatechin gallate)는 항산화, 중성지방 상승억제, 중추신경계 활성화 작용, 콜레스테롤 저하, 혈압강하, 심장혈관질환 억제에 이르는 다양한 효과가 임상시험으로 보고되고 있다. The health effects of green tea on the human body have been attracting attention worldwide, and research on bioactivity has been actively carried out in various countries around the world. EGCG (Epigallocatechin gallate) among green tea catechins has been reported to have various effects ranging from antioxidant, inhibition of triglyceride elevation, central nervous system activation, cholesterol lowering, hypotension, and cardiovascular disease inhibition.
이러한 효과를 가지는 녹차의 대표적인 카테킨 성분은, 주로 EGC(Epigallocatechin), C(Catechin), EC(Epicatechin), EGCG(Epigallocatechin gallate), ECG(Epicatechin gallate)와 이들의 광학이성질체 등이 존재한다. 일반적으로 이 5가지가 차카테킨류로 불리워지는데 이 중 EGCG가 가장 많은 양을 차지하며 생리활성 또한 가장 높은 것으로 알려져 있다. The representative catechin components of green tea having such an effect are mainly EGC (Epigallocatechin), C (Catechin), EC (Epicatechin), EGCG (Epigallocatechin gallate), ECG (Epicatechin gallate) and optical isomers thereof. In general, these five types are called tea catechins, of which EGCG is the most abundant and has the highest physiological activity.
현재 시중에 나와 있는 녹차 관련 기능성 소재들은 단순한 녹차추출물이거나 카테킨 분획물로서 이들은 모두 epigallocatechin gallate(EGCG) 이외에도 epicatechin (EC), epicatechin gallate (ECG) 및 epigallocatechin (EGC) 등 다양한 차카테킨류와 더불어 다량의 카페인을 함유하고 있지만 대체적으로 이들의 함량은 하기 표와 같이 EGCG의 함량이 60% 내외로 함유하고 있어서 실질적인효능을 가지는 유효성분인 EGCG의 함량이 충분하지 못한 단점이 있다.In addition to epigallocatechin gallate (EGCG), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and various tea catechins, as well as a large amount of caffeine The content of EGCG is about 60% as shown in the following table, and the content of EGCG, which is an effective ingredient having a substantial effect, is insufficient.
따라서 차카테킨 중 가장 우수한 기능성을 보여주는 EGCG 의 함량을 60%이상 과량 함유하는 새로운 차카테킨의 제조방법이 요구되고 있다. Therefore, there is a need for a new tea catechin production method which contains more than 60% of the EGCG content showing the best functionality among the tea catechins.
EP1077211, 출원번호 KR2001-765279호 및 출원번호 KR 2004-97357호에서는 컬럼크로마토그래피에 의해 EGCG를 고순도화하는 방법이 기재되어 있지만, 이는 EGCG를 매우 고순도로 제조하는 방법에 관한 것이지, 상기에서 살피는 바와 같이 차카테킨 혼합물의 존재중에서 EGCG의 함량을 더 증가하는 혼합물 상태로 공급되는 제품에 관한 것은 아니다. EP1077211, application number KR2001-765279 and application number KR 2004-97357 disclose a method of highly purifying EGCG by column chromatography, but this relates to a method for producing EGCG in a very high purity, But not in the form of a mixture which further increases the content of EGCG in the presence of a tea catechin mixture.
따라서 본 발명자들은 녹차잎에 자연스럽게 존재하는 성분들을 그대로 포함하면서 EGCG성분의 함량을 증가하는 새로운 방법을 제공하는 방법을 개발하여 본 발명을 완성하게 되었다.
Accordingly, the inventors of the present invention have developed a method for providing a new method for increasing the content of EGCG components, including components naturally present in green tea leaves, thereby completing the present invention.
본 발명은 녹차 유래 EGCG를 고순도 양산할 목적으로 역상 크로마토그라피와 함께 재결정법과 병행하여, 상기 역상 컬럼 크로마토그래피 전단계에서 카테킨-카페인복합물을 열수에 용해 및 급냉에 의한 석출하는 단계를 더 가짐으로써 보다 순도가 높은 EGCG를 제조할 수 있고 높은 수율로 얻을 수 있음을 알게 되어 본 발명을 완성하게 되었다.
The present invention further comprises a step of dissolving the catechin-caffeine complex in hot water and precipitating the quaternary-caffeine complex by quenching in parallel with the re-crystallization method together with the reversed phase chromatography for the purpose of mass production of green tea-derived EGCG in the reversed phase column chromatography, Can be produced at a high yield. Thus, the present invention has been completed.
본 발명은 상기의 목적을 달성하기 위하여 In order to achieve the above object,
(A) 녹차잎을 물중탕하여 차카테킨을 추출하는 단계;(A) extracting tea catechins by water bathing green tea leaves;
(B) 추출물을 농축한 후 유기용제를 이용하여 유기상으로 추출 및 농축 후 열수에 용해한 후 급냉하여 EGCG를 포함하는 카테킨-카페인 복합물을 석출 및 필터링하는 단계; 및 (B) extracting and concentrating the extract, extracting and concentrating it in an organic phase using an organic solvent, dissolving it in hot water, and then rapidly cooling to precipitate and filter the catechin-caffeine complex containing EGCG; And
(C) 상기 카테킨-카페인 복합물을 에탄올수용액을 용리액으로 하여 컬럼에 의해 분리하는 단계;(C) separating the catechin-caffeine complex by a column using an aqueous solution of ethanol as an eluent;
를 포함하여 EGCG의 함량을 고도화한 차카테킨 혼합물을 제조하는 방법을 제공한다.To provide a method for preparing a tea catechin mixture having enhanced contents of EGCG.
본 발명의 상기 제조방법에 따르면 종래의 순도 보다 훨씬 우수한 EGCG를 80~85%순도로 포함하는 차카테킨 혼합물을 제조할 수 있다. According to the preparation method of the present invention, a tea catechin mixture containing EGCG at a purity of 80 to 85%, which is far superior to the conventional purity, can be produced.
이하 본 발명의 각 단계에 대하여 설명한다.Each step of the present invention will be described below.
본 발명에 따른 물중탕에 의해 녹차잎으로부터 차카테킨을 추출하는 단계는 제한되지 않지만, 50~90℃에서 추출한다. 추출시간은 제한하지 않지만 충분히 추출할 수 있는 시간이라면 제한하지 않는다. 예를 들면 시간은 10분 내지 24시간 등 다양하게 할 수 있다. 추출 시 물중탕은 1회 또는 복수회로 추출할 수도 있으며, 복수 추출 시 이를 혼합한 후 농축하여 다음 단계로 진행할 수 있다. The step of extracting tea catechin from green tea leaves by the water bath according to the present invention is not limited, but is extracted at 50 to 90 ° C. The extraction time is not limited, but is not limited as long as it can be extracted sufficiently. For example, the time can be varied from 10 minutes to 24 hours. During the extraction, the water bath can be extracted once or plural times, and when it is extracted, it can be mixed and concentrated and then proceed to the next step.
농축은 특별히 제한되지 않지만, 물중탕 볼륨의 1~30부피%로 농축할 수 있으며 이에 제한되는 것은 아니다.Although the concentration is not particularly limited, it can be concentrated to 1 to 30% by volume of the water bath volume, but is not limited thereto.
다음 본 발명의 (B)단계에 대하여 설명한다. 본 발명에서 수상 추출물로부터 차카테킨을 유기상으로 추출하는 유기용매는 다양하게 채택할 수 있지만, 본 발명에서는 제한하지 않지만 예를 들면 에틸아세테이트가 수율면에서 가장 좋다. 추출시 에틸아세테이트는 물의 0.4 배이상, 좋게는 0.4배 내지 10배의 부피비로서 추출할 수 있지만 이에 한정하는 것은 아니다. 추출한 유기용액은 농축하여 추가 정제단계를 거치게 된다.Next, the step (B) of the present invention will be described. The organic solvent for extracting the tea catechin from the aqueous extract in the present invention into the organic phase may be variously adopted, but is not limited in the present invention, but ethyl acetate is the most preferable in terms of the yield. Ethyl acetate at the time of extraction can be extracted at a ratio of 0.4 times or more, preferably 0.4 times to 10 times, the volume of water, but is not limited thereto. The extracted organic solution is concentrated and subjected to an additional purification step.
다음 본 발명의 (C)단계에 대하여 설명한다. 본 발명의 (C)단계는 유기상을 상분리에 의해 분리한 후, 농축하고, 이를 다시 열수에 용해한 후 열수를 급냉하여 EGCG를 포함하는 카테킨-카페인 복합물을 석출 및 필터링하는 단계로서, 본 발명의 상기 (C)단계를 수행함으로서 더욱 함량 높은 EGCG를 얻을 수 있는 중간체를 얻을 수 있다. 명확하지는 않지만 이러한 열수 재용해 후 급냉하여 석출함함으로써 불필요한 불순물이 용해도에 의해 제거됨으로서 더욱 우수한 순도를 가지는 중간체를 얻을 수 있어서 좋다. Next, the step (C) of the present invention will be described. The step (C) of the present invention is a step of separating the organic phase by phase separation, concentrating it, dissolving it in hot water and then quenching the hot water to precipitate and filter the catechin-caffeine composite containing EGCG, By carrying out step (C), an intermediate can be obtained which can obtain a higher content of EGCG. It is not clear, however, that by repeating such hot water re-dissolution and quenching, unwanted impurities are removed by solubility, so that an intermediate having better purity can be obtained.
본 발명의 중간체에서 EGCG의 순도는 80~85%정도로 유지할 수 있는 놀라운 순도를 가지는 중간혼합물을 얻을 수 있었다. 이러한 순도는 HPLC를 통하여 분석할 수 있다. In the intermediate of the present invention, it was possible to obtain an intermediate mixture having an astonishing purity that can maintain the purity of EGCG at about 80 to 85%. This purity can be analyzed by HPLC.
본 발명에서 상기 열수는 50 내지 100℃ 미만이라면 크게 제한되지 않지만, 좋게는 끓는 열수를 이용하여 용해한 후, 제한되지 않지만 좋게는 20~70℃/분의 속도로 급냉하는 것이 좋다. 급냉 후에는 필요에 의해서 10분 내지 2일 정도의 시간동안 방치하는 것에의해 침전되어 석출된다. 석출된 혼합물을 분리하면 대부분이 EGCG를 포함하는 카페인등이 주 혼합물로서, 본 발명에서는 이를 카테킨-카페인복합물이라 칭한다. In the present invention, the hot water is not particularly limited as long as it is less than 50 to 100 DEG C, but preferably it is dissolved in boiling hot water and then quenched at a rate of 20 to 70 DEG C / min. After quenching, it is allowed to stand for a time of about 10 minutes to 2 days depending on necessity, and precipitation occurs. When the precipitated mixture is separated, caffeine or the like, which mainly contains EGCG, is the main mixture. In the present invention, it is referred to as a catechin-caffeine composite.
급냉은 4~30℃, 더욱 좋게는 5~20℃로 급냉하여 유지되도록 냉각하는 것이 좋지만 이에 한정하는 것은 아니다. 본 발명의 급냉 또는 급냉후 저온에서 일정기간 유지함으로서 카테킨-카페인복합체가 충분히 석출되며 석출된 상기 복합체를 필터링하여 컬럼 크로마토그래피에 의해 분리하는 단계로 분리한다.The quenching is preferably performed at a temperature of 4 to 30 ° C, more preferably 5 to 20 ° C, so as to be quenched and cooled, but the present invention is not limited thereto. The catechin-caffeine complex is sufficiently precipitated by keeping it at a low temperature for a certain period of time after quenching or quenching according to the present invention, and the precipitated complex is separated and separated by column chromatography.
다음 본 발명의 (C) 단계에 대하여 설명한다. 본 발명의 (C)단계는 상기 카테킨-카페인 복합물을 8~25중량%의 에탄올수용액을 용리액으로 하여 컬럼크로마토그래피 분리하는 단계이다. 상기 용리액의 에탄올의 함량을 조절하는 것은 불필요한 에탄올의 사용을 억제하기 위한 것으로서, 용리액은 좋게는 10%, 15% 및 20%의 에탄올 수용액을 이용하여 분별 분리한 후 이를 합쳐서 물에 의해 재결정하는 것이 좋지만 이에 한정하는 것은 아니다. 본 발명의 (C)단계에서는 특히 EGCG의 순도를 80%이상 85%로 조절함으로써 본 발명이 목적으로 하는 차카테킨 혼합물을 제조할 수 있다Next, the step (C) of the present invention will be described. The step (C) of the present invention is a step of separating the catechin-caffeine complex by column chromatography using an aqueous solution of 8 to 25% by weight of ethanol as an eluent. The amount of ethanol in the eluent is controlled to suppress the use of unnecessary ethanol. The eluent is preferably fractionated using an aqueous solution of ethanol in an amount of 10%, 15%, or 20%, and the eluent is recrystallized with water It is good, but not limited to this. In the step (C) of the present invention, particularly, the tea catechin mixture of the present invention can be prepared by controlling the purity of EGCG from 80% to 85%
상기 (C)단계에서 컬럼크로마토그래피에서 컬럼은 비인온성가교스티렌계 고정상을 이용하여 분리하는 것을 특징으로 하는데, 예를 들어 상기 고정상은 상업화된 Dianion HP-20등을 사용하는 것을 예로들 수 있고, 상기 고정상을 채택하는 경우 컬럼에 상기 고정상의 충전부피(L) 대비 시료량(kg) 값이 0.06이하일 경우 매우 우수한 분리능을 나타낸다.In the column chromatography in the step (C), the column is separated using a non-ionic crosslinked styrene-based fixed bed. For example, the stationary bed may be a commercially available Dianion HP-20 or the like. When the fixed bed is adopted, the column shows a very good separation performance when the amount of sample (kg) is 0.06 or less relative to the filling volume (L) of the stationary bed.
따라서, 본 발명에서는 종래의 유기용매로서 MC (dichloromethane)이나 MeOH (methanol) 용매계를 사용해야 하는 순상 크로마토그라피 정제방법을 채택하지 않고, 물-주정 용매계로 역상 크로마토그라피 방법으로 부분정제 하는 수단을 채택하는 것에 하나의 특징이 될 수 있다. Therefore, in the present invention, a method of partially purifying a water-alcohol solvent system by a reverse phase chromatography method is adopted without adopting a normal-phase chromatographic purification method in which MC (dichloromethane) or MeOH (methanol) solvent system is used as a conventional organic solvent It can be a feature of doing.
이하, 본 발명을 추가적으로 더 설명하면 다음과 같다.Hereinafter, the present invention will be described in further detail as follows.
본 발명에서는 (A) 녹차 분말을 물 혹은 주정(발효에탄올)로 추출, 농축한 후 초산에틸(EA)로 용매분배하는 단계, (A) extracting and concentrating the green tea powder with water or alcohol (fermented ethanol), distributing the solvent with ethyl acetate (EA)
(B) EA 층을 분리농축한 후 열수에 재용해한 후 열수를 급냉하여 EGCG를 포함하는 카테킨-카페인 복합물을 석출 및 필터링하는 단계, (B) separating and concentrating the EA layer, re-dissolving it in hot water, and then quenching the hot water to precipitate and filter the catechin-caffeine complex containing EGCG,
(C) 카테킨-카페인 복합물을 비온성 다공성충진제(Diaion HP-20 수지) 컬럼에서 물-주정 용매계로 통과하여 EGCG를 분획, 정제하는 단계를 포함한다. (C) fractionating and purifying the EGCG by passing the catechin-caffeine complex through a water-alcohol solvent system in an amorphous porous filler (Diaion HP-20 resin) column.
녹차분말을 추출하고 용매분배한 후 얻은 EA층을 충분히 농축한 다음 80℃ 온수에 녹였다가 4~20℃로 급냉하고 방치하여 두면 gum형 침전물이 가라앉는다. 이는 아마도 카페인이 카테킨류와 복합체(catechins-caffeine complex, CCC)를 형성함으로 해서 침전을 보다 용이하게 하는 듯하다. 상등액은 따라내고 침전물을 바로 컬럼으로 로딩하거나 또는 온수로 다시 녹여 Diaion HP-20수지 컬럼에 로딩(loading)하여준 다음, 5% EtOH(5배수), 15% EtOH(5배수), 20% EtOH(5배수)로 용출하여 씻어준다. 15% EtOH 및 20% EtOH 용출액을 합친 후 EA로 4회 용매분배하여 농축하면 EGCG위주분획물(순도 80~85%)이 얻어진다.
After extracting green tea powder and distributing the solvent, the EA layer obtained is sufficiently concentrated, and dissolved in hot water at 80 ° C. After quenching at 4 ~ 20 ° C, the gum-like precipitate sinks. It is likely that caffeine forms a catechins-caffeine complex (CCC), which makes it easier to precipitate. The supernatant was followed and the precipitate was either directly loaded onto the column or re-dissolved in hot water and loaded on a Diaion HP-20 resin column and then eluted with 5% EtOH (5x), 15% EtOH (5x), 20% EtOH (5 times the volume). After 15% EtOH and 20% EtOH eluate are combined, the solvent is distributed 4 times with EA and concentrated to obtain an EGCG-based fraction (purity 80-85%).
본 발명의 제조방법에 의해, 기존에 EGCG가 60%정도의 순도를 가지는 것에서 80%이상의 순도를 가지는 차카테킨을 제조할 있어서 일반 식품에 항산화첨가제 등으로 사용할 수 있다.
According to the production method of the present invention, since EGCG has a purity of about 60%, tea catechin having a purity of 80% or more can be prepared, and thus it can be used as an antioxidant additive for general food.
[실시예 1][Example 1]
녹차분말을 70℃의 물에서 6시간 추출하고 부피의 1/3으로 농축한 후 에틸아세테이트와 접촉하여 상분리에 의해 EA층에서 추출물을 회수하였다. HPLC로 확인한결과 하기 표 1과 같은 성분과 함량을 가지는 것을 확인하였다.The green tea powder was extracted with water at 70 ° C for 6 hours, concentrated to 1/3 of its volume, and then contacted with ethyl acetate to recover the extract from the EA layer by phase separation. It was confirmed by HPLC that the components and contents were as shown in Table 1 below.
[표 1][Table 1]
상기 에틸아세테이트 상을 농축한 다음 80℃ 온수에 녹였다가 10℃로 급냉하고 3일간 방치하여 두면 gum형 침전물 형태의 카페인이 카테킨류와 복합체(catechins-caffeine complex, CCC)를 형성한 침전물을 수득하였다. The ethyl acetate phase was concentrated and dissolved in hot water at 80 ° C. The resultant was rapidly cooled to 10 ° C and left standing for 3 days to obtain a precipitate in which a catechins-caffeine complex (CCC) was formed in the form of a gum-like precipitate .
이를 온수로 다시 녹여 Diaion HP-20수지 컬럼에 로딩(loading)한 다음, 5% EtOH(5배수), 15% EtOH(5배수), 25% EtOH(5배수) 및 30%EtOH(5배수)로 용출하였다.(5-fold), 15% EtOH (5-fold), 25% EtOH (5-fold) and 30% EtOH (5-fold), and then loaded onto a Diaion HP- Lt; / RTI >
상기 용출액 중에서 15% EtOH 및 20% EtOH 용출액을 합쳐서 EGCG위주 분획물을 얻었으며 이때 HPLC를 이용하여 측정한 EGCG의 순도는 84%였다.
The 15% EtOH and 20% EtOH eluates in the eluate were combined to obtain an EGCG-based fraction. The purity of EGCG measured by HPLC was 84%.
충진제 Diaion HP-20 수지의 최대 capacity 영향 평가Evaluation of maximum capacity impact of filler Diaion HP-20 resin
GL컬럼(Ф12x100cm)에 Diaion HP-20 수지를 5L로 전체적으로 채우고 아래와 같이 반복실험을 행하였다. 시료는 카페인복합체침전물(CCC PPT)을 대상으로 하였으며 이동상은 100% H2O(5L), 5% EtOH(5L), 10% EtOH(5L), 20% EtOH(5L), 25% EtOH(5L)를 순차적으로 사용하였다. 그 결과 각 용리액에 따라 얻어진 분획물의 양과 순도를 비교한 결과 하기 표 2에서 보듯이 Diaion HP-20 수지 5L 볼륨으로 충진한 경우에 시료 300g이상을 로딩하게 되면 순수 물로 용출했을 경우조차 EGCG가 충진제를 통과하여 빠져나오는 것을 보여준다. 따라서 수지충전부피(L) 대비 시료량(kg) 값이 0.06이하가 되도록 로딩하는 것이 EGCG를 분리하는데 매우 좋은 것임을 알 수 있다.A GL column (Ф12x100 cm) was filled with 5 L of Diaion HP-20 resin as a whole, and repeated experiments were carried out as follows. Samples were subjected to caffeine complex precipitate (CCC PPT) and the mobile phase was eluted with 100% H 2 O (5 L), 5% EtOH (5 L), 10% EtOH (5 L), 20% EtOH ) Were used sequentially. As a result, the amount and purity of fractions obtained according to each eluent were compared. As shown in Table 2 below, when the Diaion HP-20 resin was filled in a volume of 5 L, when more than 300 g of the sample was loaded, even when eluted with pure water, It shows passing through and exiting. Therefore, it can be understood that loading EGCG with a sample amount (kg) to be less than 0.06 with respect to the resin filling volume (L) is very good for separating EGCG.
[표 2][Table 2]
Claims (6)
(B) 추출물을 농축한 후 유기용제를 이용하여 유기상으로 추출 및 농축 후 열수에 용해한 후 급냉하여 EGCG를 포함하는 카테킨-카페인 복합물을 석출 및 필터링하는 단계; 및
(C) 상기 카테킨-카페인 복합물을 에탄올수용액을 용리액으로 하여 컬럼에 의해 분리하는 단계;
를 포함하여 제조하는 차카테킨의 추출정제방법.
(A) extracting tea catechins by water bathing green tea leaves;
(B) extracting and concentrating the extract, extracting and concentrating it in an organic phase using an organic solvent, dissolving it in hot water, and then rapidly cooling to precipitate and filter the catechin-caffeine complex containing EGCG; And
(C) separating the catechin-caffeine complex by a column using an aqueous solution of ethanol as an eluent;
≪ / RTI >
상기 (B)단계는 50~100℃미만의 열수에서 용해하고, 20~70℃/분으로 급냉하여 4~30℃에서 카테킨-카페인 복합물을 석출하는 차카테킨의 추출정제방법.
The method according to claim 1,
Wherein the step (B) is a step of dissolving in hot water at a temperature of less than 50 to 100 캜, followed by rapid cooling at 20 to 70 캜 / min to precipitate the catechin-caffeine composite at 4 to 30 캜.
상기 (C)단계의 에탄올수용액은 8~30중량%의 에탄올 수용액인 차카테킨의 추출정제방법.
3. The method of claim 2,
Wherein the ethanol aqueous solution of step (C) is an aqueous solution of ethanol in an amount of from 8 to 30% by weight.
상기 (C)단계에서 컬럼은 컬럼크로마토그래피에서의 컬럼이 비이온성가교스티렌계 고정상을 이용하여 분리하는 것을 특징으로 하는 차카테킨의 추출정제방법.
The method according to claim 1,
Wherein the column in the step (C) is separated from the column in the column chromatography by using a non-ionic crosslinking styrene-based fixed bed.
상기 고정상이 Dianion HP-20이고, 고정상의 충전부피(L) 대비 시료량(kg) 값이 0.06이하인 것을 특징으로 하는 차카테킨의 추출정제방법.
5. The method of claim 4,
Wherein the fixed bed is Dianion HP-20, and the amount of sample (kg) is not more than 0.06 with respect to the filling volume (L) of the fixed bed.
(B) 추출물을 농축한 후 유기용제를 이용하여 유기상으로 추출 및 농축 후 50~100℃미만의 열수에서 용해하고, 20~70℃/분으로 급냉하여 4~30℃에서 정치함으로써 EGCG를 포함하는 카테킨-카페인 복합물을 석출 및 필터링하는 단계; 및
(C) 상기 카테킨-카페인 복합물을 8~30wt% 범위에서 2종 이상의 농도로 에탄올수용액을 용리액으로 하여 컬럼크로마토그래피에 의해 분리하고 이들 분획을 혼합하여 분리물을 얻는 단계;
를 포함하여 제조하는 차카테킨의 추출정제방법.
(A) extracting tea catechins by water bathing green tea leaves;
(B) the extract is concentrated and extracted with an organic solvent using an organic solvent, concentrated and dissolved in hot water at a temperature of 50 to 100 ° C, quenched at 20 to 70 ° C / min and allowed to stand at 4 to 30 ° C, Precipitating and filtering the catechin-caffeine complex; And
(C) separating the catechin-caffeine complex by column chromatography using an aqueous solution of ethanol as an eluent in a concentration of 2 to 30 wt% in the range of 8 to 30 wt%, and mixing the fractions to obtain a separated product;
≪ / RTI >
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| CN105919824A (en) * | 2016-07-07 | 2016-09-07 | 北京浩奇科技有限责任公司 | EGCG-containing oily cosmetic, as well as preparation method and application thereof |
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