KR102669644B1 - Composition for improving, preventing or treating fatty liver, including milk thistle extract with increased bioavailability of silymarin - Google Patents
Composition for improving, preventing or treating fatty liver, including milk thistle extract with increased bioavailability of silymarin Download PDFInfo
- Publication number
- KR102669644B1 KR102669644B1 KR1020230082756A KR20230082756A KR102669644B1 KR 102669644 B1 KR102669644 B1 KR 102669644B1 KR 1020230082756 A KR1020230082756 A KR 1020230082756A KR 20230082756 A KR20230082756 A KR 20230082756A KR 102669644 B1 KR102669644 B1 KR 102669644B1
- Authority
- KR
- South Korea
- Prior art keywords
- milk thistle
- thistle extract
- present
- silymarin
- fatty liver
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
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- 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
-
- 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
- A23V2300/00—Processes
- A23V2300/31—Mechanical treatment
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
본 발명은 실리마린 생체이용률이 증대된 밀크씨슬 추출물의 지방간 개선, 예방 또는 치료 용도에 관한 것이다. 본 발명에서는 밀크씨슬 씨앗에 효소 처리 및 저농도 에탄올 용매 추출하여 생체이용률이 증대된 본 발명의 밀크씨슬 추출물을 제조하였고, 본 발명의 밀크씨슬 추출물이 생체이용율이 높고, 지방간 개선 예방 또는 치료에 우수한 효능을 발휘함을 확인하였다.The present invention relates to the use of milk thistle extract with increased silymarin bioavailability to improve, prevent, or treat fatty liver. In the present invention, the milk thistle extract of the present invention with increased bioavailability was prepared by treating milk thistle seeds with enzymes and extracting them with a low-concentration ethanol solvent, and the milk thistle extract of the present invention has high bioavailability and prevents or treats fatty liver disease. It was confirmed that it exhibits excellent efficacy.
Description
본 발명은 실리마린 생체이용률이 증대된 밀크씨슬 추출물의 지방간 개선, 예방 또는 치료 용도에 관한 것이다. The present invention relates to the use of milk thistle extract with increased silymarin bioavailability to improve, prevent, or treat fatty liver.
밀크씨슬(학명: Silybum marianum)은 국화과 식물로 남서유럽, 북아프리카, 아시아에서 자라며, 6월~8월에 개화하고, 1.5m 크기까지 자라는 것으로 알려져 있다. Milk thistle (scientific name: Silybum marianum ) is a plant in the Asteraceae family that grows in southwestern Europe, North Africa, and Asia. It blooms from June to August and is known to grow up to 1.5m in size.
한편, 밀크씨슬은 열매와 씨앗에 실리말린이라고 불리는 플라보노리그난 그룹을 함유하는 것으로 알려져 있는데, 연구에 따르면, 실리마린은 간세포의 세포막을 보호하고 간 기능을 개선하는 것으로 알려져 있다. 또한, 실리마린은 항산화, 항종양, 혈당강하, 항고지혈증 및 위장 보호 효능 등 다양한 약리작용을 나타내는 것으로 알려져 있다.Meanwhile, milk thistle is known to contain a group of flavonolignans called silymarin in its fruits and seeds. According to studies, silymarin is known to protect the cell membrane of liver cells and improve liver function. In addition, silymarin is known to exhibit various pharmacological effects such as antioxidant, antitumor, hypoglycemic, antihyperlipidemic, and gastrointestinal protective effects.
따라서, 상기와 같이 유용한 성분인 실리마린을 밀크씨슬로부터 추출하여 생체이용률을 높히고자 하는 연구들이 진행되고 있으나, 미진한 부분이 있다. 특히, 알코올을 용매로 사용하여 밀크씨슬로부터 실리마린을 추출하는 방법이 주로 사용되고 있으나, 실리마린 함량 대비 인체 흡수율이 낮아, 낮은 생체이용률을 보이는 문제점이 있다.Accordingly, research is being conducted to increase the bioavailability of silymarin, a useful ingredient as described above, by extracting it from milk thistle, but there is some lack of progress. In particular, the method of extracting silymarin from milk thistle using alcohol as a solvent is mainly used, but there is a problem of low bioavailability due to the low absorption rate in the human body compared to the silymarin content.
본 발명에서는 높은 실리마린 생체이용율을 보이며, 지방간 개선, 예방 또는 치료에 우수한 효능을 보이는 밀크씨슬 추출물 함유 조성물을 제공하고자 한다.The present invention seeks to provide a composition containing milk thistle extract that shows high silymarin bioavailability and shows excellent efficacy in improving, preventing, or treating fatty liver.
본 발명은 밀크씨슬 씨앗에 폴리갈락투로나아제(polygalacturonase), 셀룰라아제(cellulase), 베타글루카나아제(beta-glucanase), 및 자일라나아제(xylanase) 중 선택되는 어느 하나 이상의 효소를 첨가하여 반응시키는 단계 (a); 및 상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하는 과정으로부터 제조된 밀크씨슬 추출물을 함유하는 것을 특징으로 하는 지방간 개선용 식품 조성물을 제공한다.The present invention involves adding one or more enzymes selected from polygalacturonase, cellulase, beta-glucanase, and xylanase to milk thistle seeds. Reacting step (a); and a step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). Fatty liver, characterized in that it contains a milk thistle extract prepared from a process comprising: A food composition for improvement is provided.
또한, 본 발명은 밀크씨슬 씨앗에 폴리갈락투로나아제(polygalacturonase), 셀룰라아제(cellulase), 베타글루카나아제(beta-glucanase), 및 자일라나아제(xylanase) 중 선택되는 어느 하나 이상의 효소를 첨가하여 반응시키는 단계 (a); 및 상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하는 과정으로부터 제조된 밀크씨슬 추출물을 함유하는 것을 특징으로 하는 지방간 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides milk thistle seeds with one or more enzymes selected from polygalacturonase, cellulase, beta-glucanase, and xylanase. Adding and reacting step (a); and a step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). Fatty liver, characterized in that it contains a milk thistle extract prepared from a process comprising: A pharmaceutical composition for prevention or treatment is provided.
본 발명의 식품 조성물 또는 약학 조성물에 있어서, 상기 단계 (a)의 밀크씨슬 씨앗은 바람직하게 탈지된 밀크씨슬 씨앗인 것이 좋다. 이때, 상기 탈지된 밀크씨슬 씨앗은, 밀크씨슬 씨앗을 압착시켜 탈지시킨 것일 수 있다.In the food composition or pharmaceutical composition of the present invention, the milk thistle seeds in step (a) are preferably defatted milk thistle seeds. At this time, the defatted milk thistle seeds may be defatted by pressing milk thistle seeds.
본 발명의 식품 조성물 또는 약학 조성물에 있어서, 상기 단계 (a)의 반응은 45~55℃에서, 3~18시간 동안 반응시키는 것일 수 있다.In the food composition or pharmaceutical composition of the present invention, the reaction in step (a) may be carried out at 45 to 55 ° C. for 3 to 18 hours.
본 발명의 식품 조성물 또는 약학 조성물에 있어서, 상기 단계 (b)의 추출은 60~90℃에서, 1~8시간 동안 추출시키는 것일 수 있다.In the food composition or pharmaceutical composition of the present invention, the extraction in step (b) may be performed at 60 to 90 ° C. for 1 to 8 hours.
본 발명의 식품 조성물 또는 약학 조성물에 있어서, 상기 단계 (b)는 바람직하게 용매 추출 후, 분말화하는 과정을 더욱 포함하는 것이 좋다..In the food composition or pharmaceutical composition of the present invention, step (b) preferably further includes solvent extraction followed by powdering.
본 발명의 밀크씨슬 추출물은 실리마린 생체이용률이 높고, 지방간 개선 예방 또는 치료에 우수한 효능을 발휘한다.The milk thistle extract of the present invention has high silymarin bioavailability and exhibits excellent efficacy in preventing or treating fatty liver disease.
도 1은 효소추출 후, 30%(v/v) 에탄올을 사용하여 용매추출 시킨 본 발명 밀크씨슬 추출물 (KPMP-060, 수용성 밀크씨슬 추출물) 또는 효소추출 없이 90%(v/v)에탄올을 사용하여 용매추출 시킨 밀크씨슬 추출물 (GPMP-070, 지용성 밀크씨슬 추출물)을 쥐에게 단회 투여한 후, 얻은 실리마린 혈중농도-시간 곡선하면적으로, 본 발명 밀크씨슬 추출물의 우수한 생체이용률을 보여준다.
도 2는 본 발명 밀크씨슬 추출물 (KPMP-060)을 쥐에게 일주일간 투여한 후, 간손상을 일으키고, 측정한 혈중 AST 농도 결과로, 본 발명 밀크씨슬 추출물의 우수한 간보호 효능을 보여준다.
도 3은 본 발명 밀크씨슬 추출물 (KPMP-060)을 쥐에게 일주일간 투여한 후, 간손상을 일으키고, 측정한 혈중 ALT 농도 결과로, 본 발명 밀크씨슬 추출물의 우수한 간보호 효능을 보여준다.
도 4는 본 발명 밀크씨슬 추출물 (KPMP-060)을 쥐에게 일주일간 투여한 후, 간손상을 일으키고, 측정한 간세포의 항산화 단백질을 측정한 결과로, 본 발명 밀크씨슬 추출물의 우수한 간보호 효능을 보여준다.
도 5는 본 발명 밀크씨슬 추출물 (KPMP-060)을 쥐에게 일주일간 투여한 후, 간손상을 일으키고, 간세포를 염색하여 관찰한 결과로, 본 발명 밀크씨슬 추출물의 우수한 간보호 효능을 보여준다.
도 6은 본 발명 밀크씨슬 추출물 (KPMP-060)을 HepG2 세포에 처리하여 세포독성을 확인한 결과로, 본 발명 밀크씨슬 추출물이 세포 독성이 나타나지 않았음을 확인할 수 있다.
도 7은 본 발명 밀크씨슬 추출물 (KPMP-060)과 지방산(FFA)을 HepG2 세포에 처리(도 7의 A)하고, 본 발명 밀크씨슬 추출물의 우수한 지방 생성 억제 효능을 확인(도 7의 B)한 결과를 보여준다.
도 8은 본 발명 밀크씨슬 추출물 (KPMP-060; Sample Low, Sample High)을 처리하며, 비알콜성 지방간 동물 모델 실험을 실시하고, 쥐의 체중을 측정한 결과로, 본 발명 밀크씨슬 추출물의 우수한 지방 축적 방지 효능을 보여준다.
도 9는 본 발명 밀크씨슬 추출물 (KPMP-060; Sample Low, Sample High)을 처리하며, 비알콜성 지방간 동물 모델 실험을 실시하고, 쥐 혈청 분석을 통해 지방간 관련 바이오 마커들을 확인한 결과로, 본 발명 밀크씨슬 추출물의 우수한 지방간 개선 효능을 확인할 수 있다.
도 10은 본 발명 밀크씨슬 추출물 (KPMP-060; Sample Low, Sample High)을 처리하며, 비알콜성 지방간 동물 모델 실험을 실시하고, 얻은 쥐의 간을 염색하여 간세포 내 지방 축적을 확인한 결과로, 본 발명 밀크씨슬 추출물의 우수한 지방간 개선 효능을 확인할 수 있다.Figure 1 shows the milk thistle extract of the present invention (KPMP-060, water-soluble milk thistle extract), which was solvent-extracted using 30% (v/v) ethanol after enzyme extraction or 90% (v/v) ethanol without enzyme extraction. The area under the silymarin blood concentration-time curve obtained after administering a single dose to rats of a milk thistle extract (GPMP-070, fat-soluble milk thistle extract) extracted using a solvent, shows the excellent bioavailability of the milk thistle extract of the present invention. shows.
Figure 2 shows the excellent liver protection effect of the milk thistle extract of the present invention as a result of blood AST concentration measured after administering the milk thistle extract (KPMP-060) of the present invention to rats for one week, causing liver damage.
Figure 3 shows the excellent liver protection effect of the milk thistle extract of the present invention as a result of blood ALT concentration measured after administering the milk thistle extract (KPMP-060) of the present invention to rats for one week, causing liver damage.
Figure 4 shows the results of measuring antioxidant proteins in liver cells after administering the milk thistle extract (KPMP-060) of the present invention to rats for one week, causing liver damage, and showing the excellent liver protection of the milk thistle extract of the present invention. Shows efficacy.
Figure 5 shows the excellent liver protection effect of the milk thistle extract of the present invention as a result of administration of the milk thistle extract (KPMP-060) of the present invention to rats for one week, causing liver damage and staining of hepatocytes. .
Figure 6 shows the results of confirming cytotoxicity by treating HepG2 cells with the milk thistle extract (KPMP-060) of the present invention. It can be confirmed that the milk thistle extract of the present invention did not exhibit cytotoxicity.
Figure 7 shows that HepG2 cells were treated with the milk thistle extract (KPMP-060) of the present invention and fatty acid (FFA) (Figure 7A), and the excellent lipogenesis inhibition effect of the milk thistle extract of the present invention was confirmed (Figure 7) B) It shows one result.
Figure 8 shows the results of treating the milk thistle extract of the present invention (KPMP-060; Sample Low, Sample High), conducting a non-alcoholic fatty liver animal model experiment, and measuring the body weight of the rats, showing the milk thistle extract of the present invention shows excellent efficacy in preventing fat accumulation.
Figure 9 shows the results of processing the milk thistle extract of the present invention (KPMP-060; Sample Low, Sample High), conducting a non-alcoholic fatty liver animal model experiment, and confirming fatty liver-related biomarkers through rat serum analysis. The excellent fatty liver improvement effect of the invented milk thistle extract can be confirmed.
Figure 10 shows the results of treating the milk thistle extract of the present invention (KPMP-060; Sample Low, Sample High), conducting a non-alcoholic fatty liver animal model experiment, and staining the obtained rat liver to confirm fat accumulation in hepatocytes. , the excellent fatty liver improvement effect of the milk thistle extract of the present invention can be confirmed.
본 발명은 밀크씨슬 씨앗에 폴리갈락투로나아제(polygalacturonase), 셀룰라아제(cellulase), 베타글루카나아제(beta-glucanase), 및 자일라나아제(xylanase) 중 선택되는 어느 하나 이상의 효소를 첨가하여 반응시키는 단계 (a); 및 상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하는 과정으로부터 제조된 밀크씨슬 추출물을 함유하는 것을 특징으로 하는 지방간 개선용 식품 조성물을 제공한다.The present invention involves adding one or more enzymes selected from polygalacturonase, cellulase, beta-glucanase, and xylanase to milk thistle seeds. Reacting step (a); and a step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). Fatty liver, characterized in that it contains a milk thistle extract prepared from a process comprising: A food composition for improvement is provided.
또한, 본 발명은 밀크씨슬 씨앗에 폴리갈락투로나아제(polygalacturonase), 셀룰라아제(cellulase), 베타글루카나아제(beta-glucanase), 및 자일라나아제(xylanase) 중 선택되는 어느 하나 이상의 효소를 첨가하여 반응시키는 단계 (a); 및 상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하는 과정으로부터 제조된 밀크씨슬 추출물을 함유하는 것을 특징으로 하는 지방간 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides milk thistle seeds with one or more enzymes selected from polygalacturonase, cellulase, beta-glucanase, and xylanase. Adding and reacting step (a); and a step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). Fatty liver, characterized in that it contains a milk thistle extract prepared from a process comprising: A pharmaceutical composition for prevention or treatment is provided.
밀크씨슬은 열매와 씨앗에 실리마린을 다량 포함하는 것으로 알려져 있으며, 실리마린은 간보호, 항산화, 항종양, 혈당강하, 항고지혈증, 위장보호 효능, 지방간 개선 효능 등 다양한 기능성을 지닌 것으로 알려져 있다. 따라서, 밀크씨슬로부터 상기와 같이 유용한 실리마린을 추출하여 실리마린의 인체 흡수율을 높이기 위한 연구들이 개발되고 있으나, 높은 농도의 에탄올을 용매로 사용하여 추출시킨 밀크씨슬 추출물의 경우, 실리마린 추출 수율은 우수하지만, 지용성을 띄어 인체 흡수율이 낮은 문제점이 있다.Milk thistle is known to contain a large amount of silymarin in its fruits and seeds, and silymarin is known to have various functional properties such as liver protection, antioxidant, antitumor, hypoglycemic, antihyperlipidemic, gastrointestinal protective effect, and fatty liver improvement effect. Therefore, studies are being developed to extract useful silymarin from milk thistle as described above and increase the absorption rate of silymarin in the human body. However, in the case of milk thistle extract extracted using high concentration of ethanol as a solvent, the silymarin extraction yield is excellent. However, there is a problem in that it is fat-soluble and has a low absorption rate in the human body.
하지만, 본 발명에서는 효소 추출을 통해, 낮은 농도의 에탄올을 용매로 사용하였음에도, 높은 실리마린 함량을 가지는 수용성 밀크씨슬 추출물을 제조할 수 있었다. 또한, 본 발명에서 동물 모델 실험을 실시한 결과 본 발명의 밀크씨슬 추출물이 높은 농도의 에탄올을 용매로 사용하여 추출시킨 밀크씨슬 추출물 보다 더욱 높은 생체이용율을 보이며, 우수한 간보호 효능 및 지방간 개선 효능을 발휘함을 확인할 수 있었다.However, in the present invention, it was possible to prepare a water-soluble milk thistle extract with a high silymarin content through enzyme extraction, even though a low concentration of ethanol was used as a solvent. In addition, as a result of an animal model experiment conducted in the present invention, the milk thistle extract of the present invention shows a higher bioavailability than the milk thistle extract extracted using high concentration ethanol as a solvent, and has excellent liver protection effect and fatty liver improvement effect. It was confirmed that it works.
한편, 본 발명에서 식품 조성물은 일 예로, 츄잉껌, 캐러멜 제품, 캔디류, 빙과류, 과자류 등의 각종 식품류, 청량음료, 미네랄워터, 알코올 음료 등의 음료류, 비타민이나 미네랄 등의 건강기능성 식품류 형태로 제조된 것일 수 있다.Meanwhile, in the present invention, the food composition is, for example, manufactured in the form of various foods such as chewing gum, caramel products, candies, ice cream, and confectionery, beverages such as soft drinks, mineral water, and alcoholic beverages, and health functional foods such as vitamins and minerals. It could be.
본 발명에서 약학 조성물은 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함하여 사용 방법에 따라 바람직한 형태로 제조된 것일 수 있다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LOTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPESIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITIORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴 캅셀 중 선택되는 어느 하나일 수 있다.In the present invention, the pharmaceutical composition may be prepared in a desired form according to the method of use by further including a pharmaceutically acceptable carrier, diluent, or excipient. Examples of specific dosage forms include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, and syrups ( SYRUPS, OPHTALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), SUSPESIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITIORIES, INJECTIONS, SPIRITS, CATAPLSMA ), CAPSULES, CREAMS, TROCHES, TINCTURES, PASTES, PILLS, and soft or hard gelatin capsules.
본 발명의 약학조성물에 있어서, 투여량은 투여방법, 복용자의 연령, 성별 및 체중 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 유효성분을 기준으로 하였을 때 1일 0.0001 내지 1,000 mg/kg (체중)으로 1회 이상 경구 투여 가능하다. 다만, 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태와 의사의 처방에 의해 변화될 수 있다.In the pharmaceutical composition of the present invention, the dosage should be determined taking into account the administration method, the age, gender and weight of the recipient, and the severity of the disease. For example, based on the active ingredient, it can be administered orally at 0.0001 to 1,000 mg/kg (body weight) more than once per day. However, the above dosage is only an example for illustrative purposes, and may vary depending on the user's condition and doctor's prescription.
이하, 본 발명의 밀크씨슬 추출물 제조과정에 대해 각 단계별로 세분화하여 설명하고자 한다.Hereinafter, the production process of the milk thistle extract of the present invention will be described in detail for each step.
<단계 (a): 밀크씨슬 씨앗에 효소를 첨가하고 반응시키는 단계><Step (a): Adding enzyme to milk thistle seeds and reacting>
본 단계는 밀크씨슬 씨앗에 효소를 첨가하고 반응시키는 과정이다. 즉, 본 단계는 이후 에탄올 용매 처리과정에서 실리마린이 더욱 원활하게 추출되도록 밀크씨슬 씨앗을 효소로 전처리하는 단계이다.This step involves adding enzymes to milk thistle seeds and causing them to react. In other words, this step is to pre-treat milk thistle seeds with enzymes so that silymarin can be extracted more smoothly during the subsequent ethanol solvent treatment process.
한편, 본 단계에서 밀크씨슬 씨앗은 바람직하게 탈지된 밀크씨슬 씨앗인 것이 좋다. 탈지된 밀크씨슬 씨앗을 사용하는 경우 실리마린이 더욱 원활하게 추출되어 더욱 높은 실리마린 함량을 가진 밀크씨슬 추출물을 제조할 수 있게 된다.Meanwhile, the milk thistle seeds in this step are preferably defatted milk thistle seeds. When defatted milk thistle seeds are used, silymarin is extracted more smoothly, making it possible to produce milk thistle extract with a higher silymarin content.
탈지된 밀크씨슬 씨앗은 밀크씨슬 씨앗에 다양한 물리 화학적 방법을 수행하여 수득한 것일 수 있다. 일 예로, 밀크씨슬 씨앗을 석유 에테르로 환류 추출하여 탈지시키는 것일 수도 있고, 밀크씨슬 씨앗을 압착시켜 배유(胚乳)를 분리하여 탈지시키는 것일 수도 있다. Defatted milk thistle seeds may be obtained by performing various physical and chemical methods on milk thistle seeds. For example, milk thistle seeds may be degreased by refluxing and extracting them with petroleum ether, or milk thistle seeds may be pressed to separate the endosperm and degreased.
한편, 압착을 이용한 탈지 시 배유에 포함된 일부 전분질이 압착에 의해 제거되지 않고 껍질에 포함되어 회수될 수 있지만, 이렇게 껍질과 같이 포함되어 회수된 전분질은 본 발명의 밀크씨슬 씨앗 껍질로부터 실리마린을 추출하는데, 특별한 영향을 주는 것은 아니라서 크게 문제될 것은 없다.On the other hand, when degreasing using pressing, some of the starch contained in the endosperm may not be removed by pressing and may be recovered in the shell, but the starch contained in the shell and recovered in this way is obtained by extracting silymarin from the milk thistle seed shell of the present invention. It doesn't have any special effect on extraction, so it's not a big problem.
본 단계에서 효소는 폴리갈락투로나아제(polygalacturonase), 셀룰라아제(cellulase), 베타글루카나아제(beta-glucanase), 및 자일라나아제(xylanase) 중 선택되는 어느 하나 이상의 효소를 사용하여 반응시키는 것일 수 있는데, 바람직하게 상기 기재한 효소들을 모두 사용하는 것이 좋으며, 더욱 바람직하게는, 폴리갈락투로나아제(polygalacturonase)를 포함하는 Pectinex®Ultra SP-L 제품 (효소 활성: 3300 PGNU/g) 23~99, 셀룰라아제(cellulase)를 포함하는 Celluclast® 1.5 L 제품 (효소 활성: 700 EGU/g) 19~66, 셀룰라아제(cellulase)를 포함하는 ROHAMENT® CL 제품 (효소 활성: 15000 ECU/g) 1~6, 자일라나아제(xylanase) 및 베타글루카나아제(beta-glucanase)를 포함하는 Ultraflo® Max 제품 (베타글루카나아제 효소 활성: 700 EGU/g, 자일라나아제 효소 활성: 250 FXU/g) 1~6, 베타글루카나아제(beta-glucanase)를 포함하는 Viscozyme® L 제품 (효소활성: 100 FBG/g) 19~66의 무게 비율로 첨가하여 사용하는 것이 더욱 좋다. 하기 실시예에 따르면, 상기의 비율로 효소들을 넣고 반응시킴으로써, 높은 함량의 실리마린을 포함하면서도 생체 이용률이 높은 밀크씨슬 추출물을 제조할 수 있었다. In this step, the enzyme is reacted using one or more enzymes selected from polygalacturonase, cellulase, beta-glucanase, and xylanase. It is preferable to use all of the enzymes described above, and more preferably, Pectinex ® Ultra SP-L product containing polygalacturonase (enzyme activity: 3300 PGNU/g) 23 ~99, Celluclast ® 1.5 L product containing cellulase (enzyme activity: 700 EGU/g) 19~66, ROHAMENT ® CL product containing cellulase (enzyme activity: 15000 ECU/g) 1~ 6, Ultraflo ® Max product containing xylanase and beta-glucanase (beta-glucanase enzyme activity: 700 EGU/g, xylanase enzyme activity: 250 FXU/g) 1 ~6, It is better to use Viscozyme ® L product containing beta-glucanase (enzyme activity: 100 FBG/g) added at a weight ratio of 19 to 66. According to the following examples, by adding and reacting enzymes in the above ratio, a milk thistle extract containing a high content of silymarin and having high bioavailability could be prepared.
한편, 상기 기재한 셀룰라아제(cellulase)는 셀룰로오스를 가수분해하는 효소이다. 폴리갈락투로나아제(polygalacturonase)는 펙틴산을 가수분해하는 효소이다. 베타글루카나아제(beta-glucanase)는 베타글루칸을 가수분해하는 효소이다. 자일라나아제(xylanase)는 자일란을 가수분해하는 효소이다.Meanwhile, the cellulase described above is an enzyme that hydrolyzes cellulose. Polygalacturonase is an enzyme that hydrolyzes pectic acid. Beta-glucanase is an enzyme that hydrolyzes beta-glucan. Xylanase is an enzyme that hydrolyzes xylan.
한편, 본 단계에서 반응은 45~55℃에서, 3~18시간 동안 수행하는 것일 수 있다.Meanwhile, in this step, the reaction may be performed at 45 to 55°C for 3 to 18 hours.
<단계 (b):20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계><Step (b): extraction step by adding 20-60% (v/v) of ethanol as a solvent>
본 단계는 상기 전처리된 밀크씨슬 씨앗에 20~60%(v/v)의 에탄올을 용매로 첨가하고 추출하는 과정이다. This step is a process of adding 20 to 60% (v/v) ethanol as a solvent to the pretreated milk thistle seeds and extracting them.
일반적으로 밀크씨슬 추출물을 제조할 시에는 고농도의 에탄올을 사용하여 높은 함량으로 실리마린을 추출하는 방법이 사용된다. 하지만, 본 발명의 밀크씨슬 추출물은 20~60%(v/v)의 에탄올을 사용하여 추출하는 것에 특징이 있는데, 이를 통해, 생체이용율이 높은 수용성 실리마린 추출물을 제조할 수 있다.Generally, when producing milk thistle extract, a method of extracting silymarin at a high content using high concentration ethanol is used. However, the milk thistle extract of the present invention is characterized by extraction using 20 to 60% (v/v) ethanol, through which a water-soluble silymarin extract with high bioavailability can be prepared.
한편, 본 단계에서 추출은 60~90℃에서, 1~8시간 동안 수행하는 것일 수 있다. Meanwhile, extraction in this step may be performed at 60 to 90°C for 1 to 8 hours.
한편, 본 단계는 에탄올 용매 추출 이후, 분말화시키는 과정을 더욱 포함하는 것이 좋다. 분말화를 통해 밀크씨슬 추출물의 보관적성 및 사용적성이 향상된다.Meanwhile, this step may further include a powdering process after ethanol solvent extraction. Powdering improves the storage and usability of milk thistle extract.
상기 분말화시키는 과정은 일 예로, 동결건조를 통해 분말화시키는 것일 수 있는데, 바람직하게 동결건조 전, 여과 과정 및 농축 과정을 더욱 포함하는 것이 좋다. 여과 과정을 통해 불필요한 고형분을 제거시킬 수 있으며, 농축 과정을 통해 수분을 제거하여 분말화 과정에 도움을 줄 수 있을 뿐만 아니라, 에탄올을 제거하여 용매 잔류 문제를 해결할 수 있다.For example, the powdering process may be powdering through freeze-drying, and preferably further includes a filtration process and a concentration process before freeze-drying. Unnecessary solids can be removed through the filtration process, moisture can be removed through the concentration process to help with the powdering process, and solvent residue problems can be solved by removing ethanol.
이하, 본 발명의 내용을 하기 실시예 또는 실험예를 통하여 보다 상세하게 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 또는 실험예에만 한정되는 것은 아니고 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the contents of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples or experimental examples, but also includes modifications of the technical idea equivalent thereto.
[실시예 1: 본 발명의 밀크씨슬 추출물 제조][Example 1: Preparation of milk thistle extract of the present invention]
본 실시예에서는 하기와 같이 본 발명의 밀크씨슬 추출물을 제조하였다.In this example, the milk thistle extract of the present invention was prepared as follows.
밀크씨슬 씨앗의 껍질과 배유 (胚乳, 내부 속살)를 분리시킨 후, 회수한 밀크씨슬 씨앗 껍질 11.5 g, 하기 표 1의 효소 및 정제수를 혼합하여 68.9g의 혼합액을 제조하였다.After separating the shell and endosperm of the milk thistle seeds, 11.5 g of the recovered milk thistle seed shell, the enzyme shown in Table 1 below, and purified water were mixed to prepare a 68.9 g mixed solution.
상기 제조한 혼합액을 50℃의 온도에서 14시간 동안 1차 반응시켰다. 이후 30%(v/v) 에탄올 31.1 g을 첨가한 후, 80℃의 온도에서 4시간 동안 환류추출하며 2차 반응시켰다. 2차 반응시킨 혼합물을 25 μm의 필터로 여과시켰고, 그 통과액을 농축기를 통해 농축시켜 알코올과 수분을 제거시켰다. 이후 동결건조를 통해 본 발명의 밀크씨슬 추출물을 제조하였다.The prepared mixed solution was subjected to a primary reaction at a temperature of 50°C for 14 hours. Afterwards, 31.1 g of 30% (v/v) ethanol was added, and then refluxed and extracted at a temperature of 80°C for 4 hours to conduct a secondary reaction. The secondary reaction mixture was filtered through a 25 μm filter, and the liquid was concentrated through a concentrator to remove alcohol and moisture. Afterwards, the milk thistle extract of the present invention was prepared through freeze-drying.
[실험예 1: 본 발명 밀크씨슬 추출물의 실리마린(silymarin) 함량 분석][Experimental Example 1: Analysis of silymarin content of milk thistle extract of the present invention]
본 실험예에서는 상기 제조한 본 발명 밀크씨슬 추출물의 유용성분인 실리마린(silymarin) 함량을 분석하고자 했다. In this experimental example, the content of silymarin, a useful component, of the milk thistle extract of the present invention prepared above was analyzed.
Silymarin 6종(Silychristin, Silydianin, Silybin A, Silybin B, Isosilybin A, Isosilybin B) 각 2 mg을 메탄올(Methanol) 2ml와 혼합하여 표준용액으로 사용하고, 고속액체크로마토그래피(HPLC)를 통해 하기 표 2의 결과를 얻을 수 있었다. 2 mg each of 6 types of Silymarin (Silychristin, Silydianin, Silybin A, Silybin B, Isosilybin A, Isosilybin B) was mixed with 2 ml of methanol to use as a standard solution, and the results were obtained in Table 2 below through high-performance liquid chromatography (HPLC). results were obtained.
한편, 비교를 위해 상기 실시예 1의 제조방법을 사용하되, 효소처리 없이 30%(v/v) 에탄올을 90%(v/v) 에탄올로 대체하고, 에탄올 추출 시 85℃에서 3시간 동안 3회 반복하여 추출시킨 밀크씨슬 추출물 (대조군 1), 상기 실시예 1의 제조방법을 사용하되, 30%(v/v) 에탄올을 증류수로 대체하여 추출한 밀크씨슬 추출물 (대조군 2) 및 상기 실시예 1의 제조방법을 사용하되, 셀룰라아제(cellulase; 제품명: ROHAMENT® CL), 베타글루카나아제(β-glucanase; 제품명: Ultraflo®Max), 자일라나아제(xylanase; 제품명: Ultraflo®Max)를 사용하지 않고, 총 3종의 효소 제품만 사용하여 제조한 밀크씨슬 추출물 (대조군 3)을 대조군으로 사용하였다. Meanwhile, for comparison, the preparation method of Example 1 was used, but 30% (v/v) ethanol was replaced with 90% (v/v) ethanol without enzyme treatment, and during ethanol extraction, 3 hours at 85°C for 3 hours. Milk thistle extract extracted repeatedly (control group 1), milk thistle extract extracted using the preparation method of Example 1, but replacing 30% (v/v) ethanol with distilled water (control group 2), and the above procedure Use the manufacturing method in Example 1, but use cellulase (product name: ROHAMENT ® CL), beta-glucanase (product name: Ultraflo ® Max), and xylanase (product name: Ultraflo ® Max). Instead, milk thistle extract (control group 3) prepared using only three types of enzyme products was used as a control group.
(mg/g)Example 1
(mg/g)
(mg/g)Control group 1
(mg/g)
(mg/g)Control group 2
(mg/g)
(mg/g)Control group 3*
(mg/g)
* 대조군 3의 경우 실리마린 총합계만을 측정하였다.* For control group 3, only the total silymarin was measured.
상기 표 2를 보면, 본 발명의 실시예 1을 통해 제조한 밀크씨슬 추출물의 경우 118.15 mg/g의 실리마린을 함유하는 것을 확인할 수 있으며, 효소를 사용함에 따라 실리마린 함량이 증가한 것을 확인할 수 있다.Looking at Table 2, it can be seen that the milk thistle extract prepared through Example 1 of the present invention contains 118.15 mg/g of silymarin, and it can be seen that the silymarin content increases as the enzyme is used.
[실험예 2: 본 발명 밀크씨슬 추출물의 실리마린(silymarin)의 혈중 약물 농도 확인 실험][Experimental Example 2: Experiment to confirm blood drug concentration of silymarin of milk thistle extract of the present invention]
본 실험예에서는 본 발명 밀크씨슬 추출물의 우수한 생체이용률을 확인하고자 했다. 이를 위해, 암, 수 Sprague-Dawley rat를 이용하여 상기 제조한 대조군 1 밀크씨슬 추출물과 본 발명의 밀크씨슬 추출물의 시간에 따른 혈중 실리마린 농도를 비교하였다.In this experimental example, we sought to confirm the excellent bioavailability of the milk thistle extract of the present invention. For this purpose, the blood silymarin concentration over time of the control group 1 milk thistle extract prepared above and the milk thistle extract of the present invention were compared using male and female Sprague-Dawley rats.
1) 혈액시료 채취 방법1) How to collect blood samples
- 실험물질의 제조- Manufacture of experimental materials
대조군 1 밀크씨슬 추출물의 경우, 밀크씨슬 추출물 0.7g (실리마린 390 mg 함유)을 측량한 후, 조제용기에 부형제인 3차 멸균 증류수를 첨가하여 10 mL/kg의 투여액량으로 현탁시켰다. In the case of control group 1 milk thistle extract, 0.7 g of milk thistle extract (containing 390 mg of silymarin) was weighed, and then tertiary sterilized distilled water as an excipient was added to the preparation container and suspended at a dosage of 10 mL/kg.
본 발명의 밀크씨슬 추출물 3.3 g (실리마린 390 mg 함유)을 측량한 후, 조제용기에 부형제인 3차 멸균 증류수를 첨가하여 20 mL/kg의 투여액량으로 현탁시켰다. 본 발명의 밀크씨슬 추출물의 경우 대조군 1 밀크씨슬 추출물 보다 고형분 함량이 높아 20 mL/kg의 투여액량으로 현탁한 것이다. 다만, 투여하는 실리마린의 함량은 동일하다.After measuring 3.3 g of the milk thistle extract of the present invention (containing 390 mg of silymarin), tertiary sterilized distilled water as an excipient was added to the preparation container and suspended at a dosage of 20 mL/kg. In the case of the milk thistle extract of the present invention, the solid content was higher than that of the control group 1 milk thistle extract, so it was suspended at a dosage of 20 mL/kg. However, the amount of silymarin administered is the same.
- 실험동물 구성- Experimental animal composition
실험동물로서 수컷 SD Rat(6 주령, 180-200 g, 오리엔트바이오, 가평센터)와 암컷 SD Rat(6 주령, 130-150 g, 오리엔트바이오, 가평센터)를 사용하였고, 시험 기간 동안 케이지에서 자유롭게 물과 사료(LabDiet 5053, Nutri Int, USA)에 접근하도록 사육하였다. 온도는 22±3℃, 습도는 50±20%, 12 시간의 light/dark cycle을 유지한 상태로 실험 전 약 1주의 적응기를 거친 후 실험에 사용하였다.As experimental animals, male SD Rats (6 weeks old, 180-200 g, Orient Bio, Gapyeong Center) and female SD Rats (6 weeks old, 130-150 g, Orient Bio, Gapyeong Center) were used, and were free in cages during the test period. They were reared with access to water and food (LabDiet 5053, Nutri Int, USA). The temperature was maintained at 22 ± 3°C, the humidity was 50 ± 20%, and a 12-hour light/dark cycle was used in the experiment after an acclimatization period of about one week before the experiment.
또한, 일반증상 및 체중 증가에 이상이 없는 쥐를 대상으로 실시하였으며, 평균체중에 가까운 동물을 선발하여 각 군 평균체중이 균등하도록 무작위로 군 당 6마리로 분리하여 실험하였다.In addition, the experiment was conducted on rats with no general symptoms or weight gain, and animals with a body weight close to the average were selected and randomly divided into 6 animals per group to ensure that the average weight of each group was equal.
- 투여방법- Administration method
쥐에 시험물질을 투여하기 전 음수를 제외하고 12 시간 이상 절식을 실시한 후 일회용 주사기를 이용하여 시험물질을 위내 강제투여를 실시하였다. 대조군 1 밀크씨슬 추출물의 투여액량은 10 mL/kg으로, 본 발명의 밀크씨슬 추출물의 투여액량은 20 mL/kg으로 하고, 개체별 투여액량은 당일 측정한 체중을 기준으로 산출하여 단회 위 내에 강제 경구투여 하였다. 시험 물질 투여 1.5 시간 후에 식이를 공급하였다.Before administering the test substance to the rat, the rat was fasted for more than 12 hours, excluding drinking water, and then the test substance was forcibly administered into the stomach using a disposable syringe. Control Group 1 The administered amount of the milk thistle extract was 10 mL/kg, the administered amount of the milk thistle extract of the present invention was 20 mL/kg, and the administered amount for each individual was calculated based on the body weight measured on the day and administered as a single dose. Forced oral administration within the body. Diet was provided 1.5 hours after test substance administration.
- 혈액시료의 채취- Collection of blood samples
각 군의 모든 개체에 대하여 투여 후 채혈 시간마다 경정맥에서 헤파린 나트륨 (Sodium Heparin) (100 IU/mL)이 들어간 일회용 주사기 (1 mL 26G, 한국백신, KOR)를 이용하여 약 200 uL 이상의 혈액을 채혈하였다. 채혈시간은 투여 전 0 시간, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 3, 5, 8, 12, 24 시간으로 총 12회 채혈을 실시하였다. 채혈한 혈액은 즉시 12,000 rpm 에서 5 분간 원심분리하여 혈장을 분리한 뒤, -80℃에서 보관하였다가 전처리 전에 실온에서 해동 후 분석에 사용하였다.For all subjects in each group, approximately 200 uL or more of blood was collected from the jugular vein at each blood collection time after administration using a disposable syringe (1 mL 26G, Korea Vaccine, KOR) containing sodium heparin (100 IU/mL). did. Blood was collected 12 times in total at 0 hours, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 3, 5, 8, 12, and 24 hours before administration. The collected blood was immediately centrifuged at 12,000 rpm for 5 minutes to separate plasma, then stored at -80°C, thawed at room temperature before pretreatment, and used for analysis.
- 혈액시료의 전처리- Pretreatment of blood samples
해동한 혈장 50 μL에 100% 메탄올 (IS: naringenin 40 ng/mL 포함) 450 μL를 가하였다. 이후 vortex-mixing (2,500 rpm, 5 min)하고 10분간 원심분리 (14,000 rpm, 10 min)하였다. 상층액 1 μL를 LC-MS/MS에 주입하여 실리마린 함량을 분석하였다.450 μL of 100% methanol (IS: containing 40 ng/mL of naringenin) was added to 50 μL of thawed plasma. Afterwards, it was vortex-mixed (2,500 rpm, 5 min) and centrifuged for 10 minutes (14,000 rpm, 10 min). 1 μL of the supernatant was injected into LC-MS/MS to analyze the silymarin content.
- 기기 분석 조건- Instrument analysis conditions
UPLC 기기: 1290 Series LC system (Agilent Technologies, Palo Alto, CA, USA) UPLC instrument: 1290 Series LC system (Agilent Technologies, Palo Alto, CA, USA)
컬럼: Waters HSS T3 (1.8 um, 2.1 * 50 mm)Column: Waters HSS T3 (1.8 um, 2.1 * 50 mm)
컬럼 온도: 35℃Column temperature: 35℃
유속: 0.2 mL/minFlow rate: 0.2 mL/min
주입량: 1.0 μLInjection volume: 1.0 μL
분석시간: 16.0분Analysis time: 16.0 minutes
MS/MS 기기: 6470 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA)MS/MS instrument: 6470 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA)
2) 혈액시료 분석 결과2) Blood sample analysis results
상기 기재한 실험방법을 통해 혈액시료를 얻고, 실리마린 혈중 농도 데이터를 얻었다 (표 5~8).Blood samples were obtained through the experimental method described above, and silymarin blood concentration data were obtained (Tables 5-8).
상기 표 5~8을 보면, 본 발명의 밀크씨슬 추출물과 대조군 1 밀크씨슬 추출물을 동일한 실리마림 함량으로 투여하였음에도, 본 발명의 밀크씨슬 추출물을 투여한 경우가 더욱 높은 실리마린 혈중 농도를 보이는 것을 확인할 수 있다. 이는 본 발명 밀크씨슬 추출물이, 100% 에탄올을 사용하여 추출시킨 밀크씨슬 추출물보다, 더욱 높은 실리마린 흡수율을 보이는 것을 의미한다.Looking at Tables 5 to 8, even though the milk thistle extract of the present invention and the milk thistle extract of control group 1 were administered at the same silymarin content, the silymarin blood concentration was higher when the milk thistle extract of the present invention was administered. You can check what you see. This means that the milk thistle extract of the present invention shows a higher silymarin absorption rate than the milk thistle extract extracted using 100% ethanol.
한편, 상기 표 5~8을 바탕으로, 약동학적 파라미터를 계산하였다 (표 9~12).Meanwhile, based on Tables 5 to 8, pharmacokinetic parameters were calculated (Tables 9 to 12).
h/mL)AUC 0-t (ng·
h/mL)
h/mL)AUC 0-∞ (ng·
h/mL)
h/mL)AUC 0-t (ng·
h/mL)
h/mL)AUC 0-∞ (ng·
h/mL)
h/mL)AUC 0-t (ng·
h/mL)
h/mL)AUC 0-∞ (ng·
h/mL)
h/mL)AUC 0-t (ng·
h/mL)
h/mL)AUC 0-∞ (ng·
h/mL)
한편, 상기 표 9~12를 바탕으로, 혈중농도-시간 곡선하면적 (AUC0-∞)을 비교하였다 (도 1).Meanwhile, based on Tables 9 to 12 above, the area under the blood concentration-time curve (AUC 0-∞ ) was compared (Figure 1).
도 1을 보면, 본 발명의 밀크씨슬 추출물 (KPMP-060)과 대조군 1 밀크씨슬 추출물 (GPMP-070)이 동일한 함량으로 실리마린을 함유하고 있음에도, 본 발명의 밀크씨슬 추출물이 더욱 높은 혈중농도-시간 곡선하면적 (AUC0-∞)을 보이는 것을 확인할 수 있다. 이는 본 발명 밀크씨슬 추출물이, 100% 에탄올을 사용하여 추출시킨 밀크씨슬 추출물보다, 더욱 높은 생체이용율을 보이는 것을 의미한다.Looking at Figure 1, although the milk thistle extract of the present invention (KPMP-060) and the control group 1 milk thistle extract (GPMP-070) contain the same amount of silymarin, the milk thistle extract of the present invention has a higher blood thistle concentration. It can be seen that the area under the concentration-time curve (AUC 0-∞ ) is shown. This means that the milk thistle extract of the present invention shows a higher bioavailability than the milk thistle extract extracted using 100% ethanol.
[실험예 3: 본 발명 밀크씨슬 추출물의 간손상 방지능 평가][Experimental Example 3: Evaluation of liver damage prevention ability of milk thistle extract of the present invention]
본 실험예에서는 본 발명 밀크씨슬 추출물의 간손상 방지능을 평가하고자 했다.In this experimental example, the aim was to evaluate the liver damage prevention ability of the milk thistle extract of the present invention.
이를 위해 상기 실시예 1에서 제조한 본 발명 밀크씨슬 추출물을 쥐에게 일주일간 경구투여 한 뒤, T-BHP(Tert-Butyl hydroperoxide)를 투여하여 간손상을 유도시켰다.For this purpose, the milk thistle extract of the present invention prepared in Example 1 was orally administered to rats for a week, and then T-BHP (Tert-Butyl hydroperoxide) was administered to induce liver damage.
이후 isoflurane을 이용해 충분히 마취 한 뒤, 마취가 유지되는 상태에서 개복하고, 간조직을 채취하였다. 복대동맥에서는 전혈을 얻은 후, tube(BD SST tube, Vacutainer blood collection tube, BD)에 담아 혈청을 분리하고, 간수치 측정(AST 측정, ALT 측정)에 사용하였다.After sufficient anesthesia using isoflurane, laparotomy was performed while anesthesia was maintained, and liver tissue was collected. After obtaining whole blood from the abdominal aorta, the serum was separated in a tube (BD SST tube, Vacutainer blood collection tube, BD) and used to measure liver levels (AST measurement, ALT measurement).
1) 혈청 AST 분석 결과1) Serum AST analysis results
혈청에 존재하는 AST를 분석한 결과 (도 2), 밀크씨슬 추출물 투여 없이 T-BHP 만을 투여한 음성대조군(Negative control)은 정상대조군(Normal control)과 비교하여 혈청 AST가 유의적으로 증가한 것을 확인할 수 있었다. 또한, 본 발명의 밀크씨슬 추출물을 일주일간 경구투여 한 뒤, T-BHP를 투여하여 간손상을 유도한 실험군의 경우, 음성대조군 보다 혈청 AST 수치가 낮은 것을 확인할 수 있었다. 이는 본 발명의 밀크씨슬 추출물이 우수한 간손상 방지능이 있음을 의미한다.As a result of analyzing AST present in serum (Figure 2), the negative control group administered only T-BHP without milk thistle extract showed a significant increase in serum AST compared to the normal control group. I was able to confirm. In addition, in the experimental group in which liver damage was induced by administering T-BHP after oral administration of the milk thistle extract of the present invention for a week, it was confirmed that the serum AST level was lower than that of the negative control group. This means that the milk thistle extract of the present invention has excellent liver damage prevention ability.
2) 혈청 ALT 분석 결과2) Serum ALT analysis results
혈청에 존재하는 ALT를 분석한 결과 (도 3), 밀크씨슬 추출물 투여 없이 T-BHP 만을 투여한 음성대조군(Negative control)은 정상대조군(Normal control)과 비교하여 혈청 ALT가 유의적으로 증가한 것을 확인할 수 있었다. 또한, 본 발명의 밀크씨슬 추출물을 일주일간 경구투여 한 뒤, T-BHP를 투여하여 간손상을 유도한 실험군의 경우, 음성대조군 보다 혈청 ALT 수치가 낮은 것을 확인할 수 있었다. 이는 본 발명의 밀크씨슬 추출물이 우수한 간손상 방지능이 있음을 의미한다.As a result of analyzing ALT present in serum (Figure 3), the negative control group administered only T-BHP without milk thistle extract showed a significant increase in serum ALT compared to the normal control group. I was able to confirm. In addition, in the case of the experimental group in which liver damage was induced by administering T-BHP after oral administration of the milk thistle extract of the present invention for a week, it was confirmed that the serum ALT level was lower than that of the negative control group. This means that the milk thistle extract of the present invention has excellent liver damage prevention ability.
3) 간조직 항산화 단백질 분석3) Liver tissue antioxidant protein analysis
채취한 간조직에서 단백질을 분리하고, 항산화 단백질을 측정하였다 (도 4). 이를 통해 음성대조군(Negative control)은 정상대조군(Normal control)과 비교하여 항산화 단백질(GST, CAT, SOD)의 단백질 발현량이 감소하는 것을 확인할 수 있었다. 또한, 본 발명의 밀크씨슬 추출물을 일주일간 경구투여 한 뒤, T-BHP를 투여하여 간손상을 유도한 실험군의 경우는 항산화 단백질의 발현량이 증가하는 것을 확인할 수 있었다. 이는 본 발명의 밀크씨슬 추출물이 간의 산화스트레스 내성을 강화시켜 줄 수 있음을 의미한다. Proteins were separated from the collected liver tissue, and antioxidant proteins were measured (Figure 4). Through this, it was confirmed that the protein expression level of antioxidant proteins (GST, CAT, SOD) in the negative control group decreased compared to the normal control group. In addition, in the case of the experimental group in which liver damage was induced by administering T-BHP after oral administration of the milk thistle extract of the present invention for a week, it was confirmed that the expression level of antioxidant proteins increased. This means that the milk thistle extract of the present invention can strengthen the liver's oxidative stress resistance.
4) 간조식 변화 관찰4) Observation of tidal changes
채취한 간조직을 H&E(헤마톡실린-에오신)로 염색하여 확인한 결과 (도 5), 음성대조군(Negative control)은 정상대조군(Normal control)과 비교하여 간세포의 소실이 나타난 것을 확인할 수 있었다. 또한, 본 발명의 밀크씨슬 추출물을 일주일간 경구투여 한 뒤, T-BHP를 투여하여 간손상을 유도한 실험군의 경우, 음성대조군 보다도 간세포 소실이 덜 나타난 것을 확인할 수 있었다. 이는 본 발명의 밀크씨슬 추출물이 우수한 간손상 방지능이 있음을 의미한다.As a result of staining the collected liver tissue with H&E (hematoxylin-eosin) (Figure 5), it was confirmed that the negative control group showed loss of liver cells compared to the normal control group. In addition, in the experimental group in which liver damage was induced by administering T-BHP after oral administration of the milk thistle extract of the present invention for a week, it was confirmed that less liver cell loss occurred than the negative control group. This means that the milk thistle extract of the present invention has excellent liver damage prevention ability.
[실험예 4: 본 발명 밀크씨슬 추출물의 지방 생성 억제 효능 평가][Experimental Example 4: Evaluation of the effectiveness of the milk thistle extract of the present invention to inhibit lipogenesis]
본 실험예에서는 본 발명의 밀크씨슬 추출물(수용성 밀크씨슬 추출물, KPMP-060)의 지방 생성 억제 효능을 확인하고자 했다.In this experimental example, the aim was to confirm the effectiveness of the milk thistle extract (water-soluble milk thistle extract, KPMP-060) of the present invention to inhibit lipogenesis.
1) 세포 독성 평가1) Cytotoxicity evaluation
본 실험예에서는 지방 생성 억제 효능 확인실험을 실시하기 전, 본 발명 밀크씨슬 추출물의 세포 독성을 먼저 평가하였다.In this experimental example, before conducting an experiment to confirm the efficacy of inhibiting lipogenesis, the cytotoxicity of the milk thistle extract of the present invention was first evaluated.
구체적으로, HepG2 세포를 96-well microtiter plate에 세포농도 5×106 cells/㎖로 조절하여 100 ㎕씩 well에 넣고 수용성 밀크씨슬 추출물을을 농도별로 처리 한 다음 24시간 배양하였다. 배양액을 교환한 후 MTT labeling reagent에 electron coupling reagent를 첨가하여 준비한 MTT labeling mixture를 각 well당 10 ㎕씩(최종농도 0.5㎎/㎖) 4시간 처리한 후 550 nm 파장에서 흡광도를 이용하여 식물혼합추출물 분말의 세포독성을 측정하였다 (도 6).Specifically, HepG2 cells were adjusted to a cell concentration of 5×10 6 cells/ml in a 96-well microtiter plate, placed in 100 ㎕ each well, treated with water-soluble milk thistle extract at different concentrations, and cultured for 24 hours. After exchanging the culture medium, 10 ㎕ of MTT labeling mixture prepared by adding electron coupling reagent to the MTT labeling reagent was treated for 4 hours (final concentration 0.5 mg/ml), and then the mixed plant extract was measured using absorbance at a wavelength of 550 nm. The cytotoxicity of the powder was measured (Figure 6).
도 6을 보면, 수용성 밀크씨슬 추출물을 처리하여도, cell viability에 변화가 없는 것을 확인할 수 있다. 이는 본 발명 수용성 밀크씨슬 추출물이 세포 독성이 나타나지 않았다는 것을 의미한다.Looking at Figure 6, it can be seen that there is no change in cell viability even after treatment with the water-soluble milk thistle extract. This means that the water-soluble milk thistle extract of the present invention did not exhibit cytotoxicity.
2) 지방 생성 억제 평가2) Evaluation of inhibition of lipogenesis
본 실험예에서는 본 발명 밀크씨슬 추출물의 지방 생성 억제 효과를 확인하기 위해, HepG2 세포에 지방산 및 밀크씨슬 추출물을 처리하고, 세포 내 지방 생성 정도를 관찰하였다.In this experimental example, to confirm the inhibitory effect of the milk thistle extract of the present invention on lipogenesis, HepG2 cells were treated with fatty acids and milk thistle extract, and the degree of intracellular lipogenesis was observed.
구체적으로, HepG2 세포를 6 well-plate에 분주하고 유리 지방산(FFA)과 수용성 실리마린추출물을 24시간 처리한 후 phosphate buffered saline(PBS, Welgene, Daegu, Korea)를 이용하여 세척하고 10% formaldehyde를 이용하여 실온에서 세포를 고정하였다. 고정한 세포를 PBS로 세척하고 Oil Red O 염색시약(Sigma-Aldrich, St. Louis, MO, USA)을 넣어 실온에서 1시간 동안 염색하였다. 염색한 세포를 증류수로 세척한 다음 흡광도 측정을 위해 Isopropanol로 염색된 세포를 용출시켜 ELISA reader (Molecular Devices, CA, USA)를 이용하여 520 nm에서 흡광도를 측정하였다 (도 7).Specifically, HepG2 cells were dispensed into 6 well-plates, treated with free fatty acid (FFA) and water-soluble silymarin extract for 24 hours, washed with phosphate buffered saline (PBS, Welgene, Daegu, Korea), and treated with 10% formaldehyde. The cells were fixed at room temperature. The fixed cells were washed with PBS and stained for 1 hour at room temperature with Oil Red O staining reagent (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were washed with distilled water, and then the stained cells were eluted with isopropanol to measure absorbance, and the absorbance was measured at 520 nm using an ELISA reader (Molecular Devices, CA, USA) (Figure 7).
도 7의 A는 세포를 염색한 후 관찰한 결과를, 도 7의 B는 흡광도를 측정하여 지방 생성량을 측정한 결과를 나타낸다. 도 7을 보면, 양성 대조군 대비 본 발명의 밀크씨슬 추출물 처리군에서 지방 생성량이 적은 것을 확인할 수 있다. 이는 본 발명의 밀크씨슬 추출물이 지방 생성 억제에 효능이 있음을 의미한다.Figure 7A shows the results observed after staining the cells, and Figure 7B shows the results of measuring the amount of fat produced by measuring absorbance. Looking at Figure 7, it can be seen that the amount of fat produced was less in the milk thistle extract treatment group of the present invention compared to the positive control group. This means that the milk thistle extract of the present invention is effective in inhibiting fat production.
[실험예 5: 본 발명 밀크씨슬 추출물의 지방간 동물 모델 실험][Experimental Example 5: Fatty liver animal model experiment of milk thistle extract of the present invention]
본 실험예에서는 우수한 흡수율을 가진 본 발명의 밀크씨슬 추출물(수용성 밀크씨슬 추출물, KPMP-060)이 대조군 1 밀크씨슬 추출물 (지용성 밀크씨슬 추출물, GPMP-070) 보다 지방간에 더욱 우수한 효능을 보일 수 있는지 확인하고자 했다.In this experimental example, the milk thistle extract of the present invention (water-soluble milk thistle extract, KPMP-060), which has excellent absorption rate, had a better effect on fatty liver than the control group 1 milk thistle extract (oil-soluble milk thistle extract, GPMP-070). I wanted to see if I could show it.
이를 위해, 생후 6주령 된 수컷 쥐(C56BL/6)에게 비알콜성 지방간(Nonalcoholic Fatty Liver Disease, NAFLD)을 유발시키며, 밀크씨슬 추출물을 처리하고, 쥐의 체중, 혈청 내 지표, 간 조직의 지방 축적량을 확인하였다.For this purpose, Nonalcoholic Fatty Liver Disease (NAFLD) was induced in 6-week-old male mice (C56BL/6), treated with milk thistle extract, and the weight of the mice, serum indicators, and liver tissue were measured. The amount of fat accumulation was confirmed.
1) 지방간 동물모델 실험 방법1) Fatty liver animal model experiment method
생후 6주령 된 수컷 쥐(C56BL/6)에게 고지방 식이(60% kcal fat diet)와 20% fructose가 함유된 음수를 병행 섭취시킴으로써 지방간을 유발시키고, 밀크씨슬 추출물을 처리하여 효능을 확인하였다. Fatty liver was induced in 6-week-old male mice (C56BL/6) by simultaneously consuming a high-fat diet (60% kcal fat diet) and drinking water containing 20% fructose, and the efficacy was confirmed by treatment with milk thistle extract.
정상 대조군(Normal diet control)에서는 정상 식이(AIN-93G)를 사용했으며, 비알콜성 지방간 유발군은 고지방 식이(60% kcal fat diet)와 20% fructose 가 함유된 음수를 병행 섭취시킴으로써 지방간을 유발하였다. 식이와 음수는 9주간 자유롭게 섭취하게 하고 시험물질인 수용성 실리마린(65mg/kg, 130mg/kg)과 대조약물인 지용성 실리마린(13.5mg/kg) 투여를 식이유발과 함께 진행하였고 1일 1회 경구 투여하였다. 정상 대조군(Normal diet control)에서는 증류수만을 경구 투여하였다 (표 13 참조).The normal diet control group used a normal diet (AIN-93G), and the non-alcoholic fatty liver disease group induced fatty liver disease by consuming a high-fat diet (60% kcal fat diet) and drinking water containing 20% fructose. did. Diet and drinking water were freely consumed for 9 weeks, and the test substance, water-soluble silymarin (65 mg/kg, 130 mg/kg), and the control drug, fat-soluble silymarin (13.5 mg/kg) were administered along with dietary induction, and were administered orally once a day. did. In the normal diet control, only distilled water was administered orally (see Table 13).
(60% kcal fat)
+
20% Fructose 음수High Fat diet
(60% kcal fat)
+
20% Fructose Negative
한편, 양성 대조군(Positive control)에서 지용성 밀크씨슬 추출물은 13.5mg/kg을 투여하였고, 저농도 실시군(Sample Low)에서 수용성 밀크씨슬 추출물은 65mg/kg을 투여하였는데, 각 추출물에 함유된 실리마린 함량을 고려하여, 투여한 것이다. 즉, 양성 대조군(Positive control)과 저농도 실시군(Sample Low)에서 투여되는 실리마린의 함량은 동일하다.Meanwhile, in the positive control group, 13.5 mg/kg of fat-soluble milk thistle extract was administered, and in the low concentration group (Sample Low), 65 mg/kg of water-soluble milk thistle extract was administered. The silymarin contained in each extract It was administered considering the content. That is, the amount of silymarin administered in the positive control group and the low concentration group (Sample Low) was the same.
2) 체중 측정2) Weight measurement
지방간 동물모델 실험이 진행되는 동안 일주일 간격으로 실험 동물의 체중을 측정하였다 (도 8).While the fatty liver animal model experiment was in progress, the body weight of the experimental animals was measured at weekly intervals (FIG. 8).
도 8을 보면, 양성 대조군(Positive control) 대비, 수용성 밀크씨슬 추출물 처리군(Sample Low, Sample High)에서 체중이 더욱 낮은 것을 확인할 수 있다. 이는 본 발명의 수용성 밀크씨슬 추출물이 지방 축적 방지에 효능이 있음을 의미한다.Looking at Figure 8, it can be seen that body weight was lower in the water-soluble milk thistle extract treated group (Sample Low, Sample High) compared to the positive control. This means that the water-soluble milk thistle extract of the present invention is effective in preventing fat accumulation.
3) 혈청 내 지표 변화3) Changes in indicators in serum
9주간의 지방간 동물모델 실험이 끝난 후, 실험동물로부터 얻은 혈액을 3000rpm에서 15분간 원심분리하여 혈청을 분리하고, 지방간 관련 바이오 마커들을 측정하였다. GOT(Glutamic oxaloacetic transaminase), GPT(Glutamic pyruvic transaminase), 총 콜레스테롤(TC), 중성지방(TG), HDL(High density lipoprotein cholesterol) 수치는 분리한 혈청을 혈액분석기와 kit를 이용하여 측정하였다. LDL(Low density lipoprotein cholesterol) 수치는 Freidewald 등의 방법에 따라 LDL cholesterol = total cholesterol - HDL cholesterol - TG/5 식을 이용하여 산출하였다 (도 9). After the 9-week fatty liver animal model experiment was completed, blood obtained from the experimental animals was centrifuged at 3000 rpm for 15 minutes to separate serum, and fatty liver-related biomarkers were measured. Glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), total cholesterol (TC), triglyceride (TG), and high density lipoprotein cholesterol (HDL) levels were measured in separated serum using a blood analyzer and kit. LDL (low density lipoprotein cholesterol) levels were calculated using the formula LDL cholesterol = total cholesterol - HDL cholesterol - TG/5 according to the method of Freidewald et al. (Figure 9).
도 9를 보면, 본 발명의 수용성 밀크씨슬 추출물 처리군(Sample Low, Sample High)에서 GOT, GPT, 중성지방(TG), LDL 지표가 낮게 나오는 것을 확인할 수 있다. 이는 본 발명의 수용성 밀크씨슬 추출물이 지방간에 우수한 효능이 있음을 의미한다.Looking at Figure 9, it can be seen that GOT, GPT, triglyceride (TG), and LDL indicators are low in the water-soluble milk thistle extract treatment group (Sample Low, Sample High) of the present invention. This means that the water-soluble milk thistle extract of the present invention has excellent efficacy against fatty liver.
4) 간 조직의 지방 축적 분석4) Analysis of fat accumulation in liver tissue
실험 종료 후 실험동물을 희생하여 마우스에서 분리한 간 조직을 10% phosphate-buffered formalin에서 하루 이상 처리하여 고정하고, 12시간 이상 흐르는 물에서 formalin을 세척한 후, 60% ethanol, 70% ethanol, 80% ethanol 90% ethanol, 95% ethanol, 100% ethanol을 각각 1시간씩 처리하여 탈수시켰다. Xylen을 처리하여 1시간씩 3번의 투명화 과정 후, paraffin을 1시간씩 2번 처리하는 침투과정을 실시하였다. 포매과정을 거쳐 약 2㎛의 두께로 박절하여 slide 위에 조직을 얹고 건조시킨 후, hematoxylin-eosin 염색을 실시하였다. Slide의 물기를 제거하고, mounting medium을 처리한 후, cover glass를 덮어 마무리하였다. 이후, 간의 지방조직 분석을 Axio observer Z1 microscope (Carl Zeiss Inc., Gottingen, Germany)을 이용하여 실시하였다 (도 10).After completing the experiment, the experimental animals were sacrificed and the liver tissue isolated from the mouse was fixed by treating it in 10% phosphate-buffered formalin for more than a day. After washing the formalin in running water for more than 12 hours, it was washed with 60% ethanol, 70% ethanol, and 80% ethanol. % ethanol 90% ethanol, 95% ethanol, and 100% ethanol were each treated for 1 hour to dehydrate. After the transparency process was performed three times for 1 hour each using xylen, a penetration process was performed using paraffin twice for one hour each. After the embedding process, the tissue was sectioned to a thickness of approximately 2㎛, the tissue was placed on a slide, dried, and hematoxylin-eosin staining was performed. After removing the moisture from the slide, processing the mounting medium, and finishing it with a cover glass. Afterwards, liver fat tissue analysis was performed using an Axio observer Z1 microscope (Carl Zeiss Inc., Gottingen, Germany) (FIG. 10).
도 10을 보면, 고지방+과당 음수를 투여한 대조군(Negative Control, Positive Control)에서 주변부의 하얀색으로 염색된 지방 알갱이가 관찰되며, 지질에 의해 핵이 밀려나 있는 전형적인 지방간 형태를 확인할 수 있다. 반면, 본 발명의 수용성 밀크씨슬 추출물(Sample Low, Sample High)을 처리한 경우에는 주변부에 작은 지방 입자만이 관찰되는 것을 확인할 수 있다. 상기와 같은 결과는 본 발명의 수용성 밀크씨슬 추출물이 간세포 내 지방축적을 감소시키며 지방간에 우수한 효능을 보였음을 의미한다.Looking at Figure 10, in the control group (Negative Control, Positive Control) administered high-fat + fructose negative water, fat granules stained white at the periphery are observed, and a typical form of fatty liver with the nucleus pushed out by lipids can be confirmed. On the other hand, when treated with the water-soluble milk thistle extract (Sample Low, Sample High) of the present invention, it can be seen that only small fat particles are observed in the peripheral area. The above results mean that the water-soluble milk thistle extract of the present invention reduced fat accumulation in hepatocytes and showed excellent efficacy against fatty liver.
Claims (7)
상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하는 과정으로부터 제조된 밀크씨슬 추출물을 함유하는 것을 특징으로 하는 지방간 개선용 식품 조성물.
Adding polygalacturonase, cellulase, beta-glucanase, and xylanase to defatted milk thistle seeds to react (a); and
Improving fatty liver, characterized in that it contains a milk thistle extract prepared from a process including step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). For food composition.
상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하는 과정으로부터 제조된 밀크씨슬 추출물을 함유하는 것을 특징으로 하는 지방간 예방 또는 치료용 약학 조성물.
Adding polygalacturonase, cellulase, beta-glucanase, and xylanase to defatted milk thistle seeds to react (a); and
Fatty liver prevention, characterized in that it contains a milk thistle extract prepared from a process including step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). or therapeutic pharmaceutical compositions.
상기 탈지된 밀크씨슬 씨앗은,
밀크씨슬 씨앗을 압착시켜 탈지시킨 것을 특징으로 하는 지방간 개선용 식품 조성물.
According to paragraph 1,
The defatted milk thistle seeds,
A food composition for improving fatty liver, characterized in that milk thistle seeds are pressed and defatted.
상기 탈지된 밀크씨슬 씨앗은,
밀크씨슬 씨앗을 압착시켜 탈지시킨 것을 특징으로 하는 지방간 예방 또는 치료용 약학 조성물.
According to paragraph 2,
The defatted milk thistle seeds,
A pharmaceutical composition for preventing or treating fatty liver disease, characterized in that milk thistle seeds are pressed and defatted.
상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하여 밀크씨슬 추출물을 제조하는 과정을 포함하는 것을 특징으로 하는 지방간 개선용 식품 조성물의 제조방법.
Adding polygalacturonase, cellulase, beta-glucanase, and xylanase to defatted milk thistle seeds to react (a); and
Improving fatty liver disease, comprising the step (b) of extracting milk thistle extract by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). Method for producing a food composition.
상기 단계 (a) 후, 20~60%(v/v)의 에탄올을 용매로 첨가하여 추출하는 단계 (b);를 포함하여 밀크씨슬 추출물을 제조하는 과정을 포함하는 것을 특징으로 하는 지방간 예방 또는 치료용 약학 조성물의 제조방법.
Adding polygalacturonase, cellulase, beta-glucanase, and xylanase to defatted milk thistle seeds to react (a); and
Fatty liver prevention, comprising a process of preparing a milk thistle extract, including step (b) of extracting by adding 20 to 60% (v/v) of ethanol as a solvent after step (a). Or a method for producing a therapeutic pharmaceutical composition.
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Citations (5)
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|---|---|---|---|---|
| DE10016449A1 (en) * | 2000-03-29 | 2001-10-04 | Council Scient Ind Res | Production of silymarin from Silybum marianum seeds includes defatting of powdered seeds with hydrocarbon solvent, extraction with acetonitrile and purification using dichloromethane |
| KR20100126272A (en) * | 2007-12-23 | 2010-12-01 | 유로메드 에스.에이. | Novel milk thistle extract, method for the production, and use |
| KR20130102305A (en) | 2012-03-07 | 2013-09-17 | 주식회사 케어사이드 | Composition for liver protection and improving liver function and functional food comprising thererof |
| KR20160115461A (en) * | 2015-03-27 | 2016-10-06 | 전라남도 | A high efficient method for extracting silymarin from cirsium pendulum |
| KR20200057006A (en) | 2017-09-22 | 2020-05-25 | 유로메드, 에스.에이. | Improved solubility of milk thistle extract |
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| DE10016449A1 (en) * | 2000-03-29 | 2001-10-04 | Council Scient Ind Res | Production of silymarin from Silybum marianum seeds includes defatting of powdered seeds with hydrocarbon solvent, extraction with acetonitrile and purification using dichloromethane |
| KR20100126272A (en) * | 2007-12-23 | 2010-12-01 | 유로메드 에스.에이. | Novel milk thistle extract, method for the production, and use |
| KR20130102305A (en) | 2012-03-07 | 2013-09-17 | 주식회사 케어사이드 | Composition for liver protection and improving liver function and functional food comprising thererof |
| KR20160115461A (en) * | 2015-03-27 | 2016-10-06 | 전라남도 | A high efficient method for extracting silymarin from cirsium pendulum |
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