KR102483627B1 - Manufacturing method of the strawberry stem extracts and Anti-oxidant and anti-inflammatory inner beauty composition using the same - Google Patents
Manufacturing method of the strawberry stem extracts and Anti-oxidant and anti-inflammatory inner beauty composition using the same Download PDFInfo
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- KR102483627B1 KR102483627B1 KR1020210179083A KR20210179083A KR102483627B1 KR 102483627 B1 KR102483627 B1 KR 102483627B1 KR 1020210179083 A KR1020210179083 A KR 1020210179083A KR 20210179083 A KR20210179083 A KR 20210179083A KR 102483627 B1 KR102483627 B1 KR 102483627B1
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- extract
- strawberry
- measured
- equation
- strawberry stem
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
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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
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/92—Oral administration
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Public Health (AREA)
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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Microbiology (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Dermatology (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Description
본 발명은 딸기 꼭지 추출물을 이용한 항산화 및 항염증 이너뷰티 조성물 및 이를 제조하는 방법에 관한 것이다.The present invention relates to an antioxidant and anti-inflammatory inner beauty composition using strawberry stem extract and a method for preparing the same.
재배 딸기(Fragaria ananassa Duch.)는 장미과에 속하는 다년생 식물로 재배 기간이 길고 노동력이 많이드는 작물에 속하지만 저온에서 생육이 가능하며 저가온 시설재배로 11월부터 5월까지 수확할 수 있다. 재배딸기에는 플라보노이드(flavonid)류로 퀘르세틴-7-자일로사이드(quercetin-7-xyloside), 프라가린(fragarin), 프로시아니딘 B3(procyanidin B3), 1-O-p-쿠마로일글루코스(1-O-p-coumaroylglucose), 2-헵탄올(2-heptanol) 또는 엘라직산(ellagic acid) 등을 포함하는 것으로 알려져 있다. 이에 딸기를 이용한 항염, 항암, 신장질환 등 질환 관련 많은 연구들이 다수 진행되고 있다. 또한, 향기와 색상이 우수하여 현재 잼, 시럽, 주스 등의 제조 원료로 많이 사용되고 있으며, 이를 이용한 가공품 가치도 높아 관련 연구들도 많이 진행되어 있는 상태이다. 딸기의 꼭지(꽃받침) 부분은 섭취 전 또는 가공 전에 대부분 버려지는 부산물로서, 가공에 소요되는 딸기 원료량은 2008년 기준으로 약 1만톤(냉동딸기 포함) 이상이고, 이중 딸기 꼭지가 차지하는 비율이 약 5%이기 때문에 거의 500톤 가량이 버려지며, 이는 매우 큰 자원 낭비이다. 딸기 꼭지의 생리활성 강화 유효성분을 딸기 꼭지에는 인체에 유익한 성분을 다량 포함하고 있는데, 이를 활용한 기술 및 제품 범위가 매우 좁은 실정이다.Cultivated strawberry (Fragaria ananassa Duch.) is a perennial plant belonging to the Rosaceae family that has a long cultivation period and is a labor-intensive crop, but can be grown at low temperatures and can be harvested from November to May through low-temperature facility cultivation. Cultivated strawberries contain flavonoids such as quercetin-7-xyloside, fragarin, procyanidin B3, and 1-O-p-coumaroylglucose. ), 2-heptanol or ellagic acid. As a result, many studies on diseases such as anti-inflammatory, anti-cancer, and kidney diseases using strawberries are being conducted. In addition, because of its excellent fragrance and color, it is currently used as a raw material for manufacturing jams, syrups, juices, etc. The stem (calyx) of strawberries is a by-product that is mostly discarded before consumption or processing. As of 2008, the amount of strawberry raw materials required for processing was about 10,000 tons (including frozen strawberries), and the proportion of strawberry stems was about 5 %, so nearly 500 tons are wasted, which is a very large waste of resources. Strawberry stems contain a large amount of active ingredients that enhance the physiological activity of strawberry stems, which are beneficial to the human body, but the range of technologies and products using them is very narrow.
본 발명은 섭취 전 /또는 가공 전에 딸기 과실로부터 제거되어 버려지는 딸기 꼭지를 가공하여 항산화성 및 항염증성 유효성분을 추출하여 이를 이용한 이너뷰티 조성물을 제공하고자 한다.The present invention is to extract antioxidant and anti-inflammatory active ingredients by processing strawberry stems that are removed and discarded from strawberry fruits before consumption and/or processing, and provide an inner beauty composition using the same.
상술한 과제를 해결하기 위한, 본 발명의 항산화성 및 항염성 이너뷰티 조성물은 딸기 꼭지 추출물을 유효성분으로 포함하며, 상기 딸기 꼭지 추출물은 열수 추출물 또는 탄소수 1 ~ 4의 저급 알코올 추출물이다.In order to solve the above problems, the antioxidant and anti-inflammatory inner beauty composition of the present invention contains strawberry stem extract as an active ingredient, and the strawberry stem extract is a hot water extract or a lower alcohol extract having 1 to 4 carbon atoms.
또한, 본 발명은 딸기 꼭지 추출물의 제조방법에 관한 것으로서, 딸기 꼭지를 세척 및 건조시킨 후, 분쇄하여 딸기 꼭지 분말을 준비하는 1단계; 기 딸기 꼭지 분말 및 물을 1 : 8 ~ 12 중량비로 혼합한 후, 95 ~ 110℃에서 2 ~ 4시간 동안 환류 냉각 추출한 후, 냉각 추출액을 10 ~ 35℃에서 18 ~ 30 시간 동안 방치하는 2단계; 단계를 수행한 추출액을 여과하여 상등액과 침전물을 분리하는 3단계; 및 상등액을 진공감압농축을 수행하여 농축물을 수득한 후, 동결건조를 수행하여 열수 추출물을 수득하는 4단계;를 포함하는 공정을 수행할 수 있다.In addition, the present invention relates to a method for producing a strawberry stem extract, comprising the steps of preparing strawberry stem powder by washing and drying strawberry stems and then pulverizing them; Strawberry stalk powder and water are mixed in a weight ratio of 1:8 to 12, extracted by reflux cooling at 95 to 110 °C for 2 to 4 hours, and then the cooled extract is left at 10 to 35 °C for 18 to 30 hours. ; Step 3 of separating the supernatant and the precipitate by filtering the extract obtained by performing the step; and 4 steps of subjecting the supernatant to vacuum concentration to obtain a concentrate, followed by lyophilization to obtain a hot water extract.
또한, 본 발명은 딸기 꼭지 추출물의 제조방법은 딸기 꼭지를 세척 및 건조시킨 후, 분쇄하여 딸기 꼭지 분말을 준비하는 1단계; 상기 딸기 꼭지 분말 및 탄소수 1 ~ 4의 저급 알코올 수용액을 혼합한 후, 20 ~ 30℃에서 18 ~ 30시간 동안 교반을 수행하는 2단계; 2단계를 수행한 추출액을 여과하여 상등액과 침전물을 분리하는 3단계; 상등액을 진공감압농축을 수행하여 농축물을 수득한 후, 동결건조를 수행하여 알코올 추출물을 수득하는 4단계;를 포함하는 공정을 수행하여 제조할 수도 있다.In addition, the present invention is a method for producing a strawberry stem extract comprising the first step of preparing strawberry stem powder by washing and drying strawberry stems and then pulverizing them; A second step of mixing the strawberry stem powder and an aqueous solution of lower alcohol having 1 to 4 carbon atoms, followed by stirring at 20 to 30 ° C. for 18 to 30 hours; Step 3 of separating the supernatant and the precipitate by filtering the extract obtained in
본 발명의 딸기 꼭지 추출물은 낮은 세포독성을 가지고, 다량의 폴리페놀을 함유하고 있는 바, 전자공여능 활성 및 ABTS(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 라디칼 소거능이 우수하여 항산화성이 우수하며, NO 생성 억제 및 iNOS(Nitric oxide synthase)와 COX-2(Prostaglandin-endoperoxide synthase 2) 단백질 발현을 억제하여 항염증성이 우수한다.The strawberry stem extract of the present invention has low cytotoxicity and contains a large amount of polyphenols, so it has electron donating activity and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity. It has excellent antioxidant properties and anti-inflammatory properties by suppressing NO production and iNOS (nitric oxide synthase) and COX-2 (Prostaglandin-endoperoxide synthase 2) protein expression.
도 1은 실험예 1에서 실시한 딸기 꼭지 열수 추출물 및 에탄올 추출물의 전자공여능(DPPH assay, 1-1-diphenyl-2-picryl-hydrazyl) 측정 결과이다.
도 2은 실험예 1에서 실시한 딸기 꼭지 열수 추출물 및 에탄올 추출물의 ABTS(2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) 라디칼 소거능 측정 결과이다.
도 3은 실험예 2에서 실시한 세포 독성(또는 세포 생존율) 측정 결과이다.
도 4는 실험예 3에서 실시한 NO 생성 저해 측정 결과이다.
도 5는 실험예 4에서 실시한 iNOS 단백질 발현 저해 측정 결과이다.
도 6은 실험예 4에서 실시한 COX-2 단백질 발현 저해 측정 결과이다.1 is a result of measuring the electron donating ability (DPPH assay, 1-1-diphenyl-2-picryl-hydrazyl) of strawberry stem hot water extract and ethanol extract conducted in Experimental Example 1.
2 is a result of measuring ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity of strawberry stem hot water extract and ethanol extract conducted in Experimental Example 1.
3 is a result of measuring cytotoxicity (or cell viability) performed in Experimental Example 2.
4 is a result of NO production inhibition measurement conducted in Experimental Example 3.
5 is a result of measuring inhibition of iNOS protein expression conducted in Experimental Example 4.
6 is a result of COX-2 protein expression inhibition measurement conducted in Experimental Example 4.
본 발명에서 사용하는 용어인 "이너뷰티(inner beauty)"는 내부에서부터 건강한 피부를 가꾼다는 의미로서, 먹는 화장품을 통틀어 이르는 말이다.The term "inner beauty" used in the present invention means to take care of healthy skin from the inside, and refers to cosmetics to be eaten.
이하에서 본 발명의 이너뷰티 소재인 딸기 꼭지 추출물 제조하는 방법을 통해서 본 발명을 더욱 자세하게 설명한다.Hereinafter, the present invention will be described in more detail through a method for manufacturing strawberry stem extract, which is the inner beauty material of the present invention.
딸기 꼭지는 꽃받침이라고 불리며, 이는 5 ~ 6개의 녹색 잎으로 구성되며 피침형으로 긴 타원형보다 좁고 양끝이 뾰족한 모양이다. 딸기의 과실부분에서 분리한 꼭지 부분을 세척하여 열풍 건조한 후 파쇄하여 추출물 제조에 사용하였다.Strawberry stems are called calyxes, which consist of 5 to 6 green leaves and are lanceolate, narrower than long ovals and pointed at both ends. The stem part separated from the fruit part of the strawberry was washed, dried with hot air, and then crushed and used to prepare the extract.
본 발명의 딸기 꼭지 추출물은 딸기 꼭지 열수 추출물 및/또는 딸기 꼭지 알코올 추출물일 수 있다.The strawberry stem extract of the present invention may be a strawberry stem hot water extract and/or a strawberry stem alcohol extract.
상기 딸기 꼭지 열수 추출물은 딸기 꼭지를 세척 및 건조시킨 후, 분쇄하여 딸기 꼭지 분말을 준비하는 1단계; 상기 딸기 꼭지 분말 및 물을 혼합한 후, 95 ~ 110℃에서 2 ~ 4시간 동안 환류 냉각 추출한 후, 냉각 추출액을 10 ~ 35℃에서 18 ~ 30시간 동안, 바람직하게는 20 ~ 26시간 동안 방치하는 2단계; 2단계를 수행한 추출액을 여과하여 상등액과 침전물을 분리하는 3단계; 및 상등액을 진공감압농축을 수행하여 농축물을 수득한 후, 동결건조를 수행하여 열수 추출물을 수득하는 4단계;를 포함하는 공정을 수행하여 제조할 수 있다. 그리고, 상기 상기 3단계는 1 ~ 3회 반복 수행하여 제조할 수 있다.The strawberry stem hot water extract is prepared by washing and drying the strawberry stems and then pulverizing to prepare strawberry stem powder; After mixing the strawberry stem powder and water, reflux cooling extraction at 95 ~ 110 ℃ for 2 ~ 4 hours, and then the cooled extract is left at 10 ~ 35 ℃ for 18 ~ 30 hours, preferably for 20 ~ 26
열수 추출물 제조공정에 있어서, 1단계에서 딸기 꼭지 분말 및 물을 1:7 ~ 14 중량비로, 바람직하게는 1 : 8 ~ 12 중량비로, 더욱 바람직하게는 딸기 꼭지 분말 및 물을 1 : 9 ~ 11 중량비로 혼합하는 것이 좋으며, 이때, 물 혼합량이 8 중량비 미만이거나, 12 중량부를 초과하면 용매 용량대비 추출효율이 낮아지는 문제가 있을 수 있으므로 상기 범위 내로 혼합사용하는 것이 좋다.In the hot water extract manufacturing process, in the first step, strawberry stem powder and water are mixed in a weight ratio of 1:7 to 14, preferably 1:8 to 12, and more preferably strawberry stem powder and water are mixed in a weight ratio of 1:9 to 11 It is good to mix in a weight ratio, and at this time, if the water mixture is less than 8 parts by weight or exceeds 12 parts by weight, there may be a problem of lowering the extraction efficiency compared to the solvent capacity, so it is good to mix and use within the above range.
또한, 딸기 꼭지 알코올 추출물은 딸기 꼭지를 세척 및 건조시킨 후, 분쇄하여 딸기 꼭지 분말을 준비하는 1단계; 상기 딸기 꼭지 분말 및 알코올 수용액을 혼합한 후, 20 ~ 30℃에서, 바람직하게는 22 ~ 27℃에서, 18 ~ 30시간 동안 교반을 수행하는 2단계; 2단계를 수행한 추출액을 여과하여 상등액과 침전물을 분리하는 3단계; 및 상등액을 진공감압농축을 수행하여 농축물을 수득한 후, 동결건조를 수행하여 알코올 추출물을 수득하는 4단계;를 포함하는 공정을 수행하여 제조할 수 있다.In addition, the strawberry stem alcohol extract includes a first step of preparing strawberry stem powder by washing and drying strawberry stems and then pulverizing them; A second step of mixing the strawberry stalk powder and the aqueous alcohol solution and stirring at 20 to 30 ° C., preferably at 22 to 27 ° C., for 18 to 30 hours; Step 3 of separating the supernatant and the precipitate by filtering the extract obtained in
알코올 추출물 제조공정의 1단계에서 상기 알코올 수용액은 탄소수 1 ~ 4의 저급 알코올 수용액을 사용할 수 있고, 바람직하게는 에탄올 수용액일 수 있으며, 더욱 바람직하게는 65 ~ 80 부피% 농도의 에탄올 수용액을 사용할 수 있다. 에탄올 수용액 사용시 에탄올 수용액의 농도가 65 부피% 미만이면 추출액 내 생리활성 유효성분 함량이 낮을 수 있고, 농도가 80 부피%를 초과하면 용매 농도 대비 추출 효율이 높지 않는 문제가 있을 수 있으므로 상기 부피% 농도의 에탄올 수용액을 사용하는 것이 바람직하다.In the first step of the alcohol extract manufacturing process, the aqueous alcohol solution may use an aqueous solution of a lower alcohol having 1 to 4 carbon atoms, preferably an aqueous ethanol solution, and more preferably an aqueous ethanol solution having a concentration of 65 to 80% by volume. there is. When using an aqueous ethanol solution, if the concentration of the aqueous ethanol solution is less than 65% by volume, the content of the physiologically active active ingredient in the extract may be low, and if the concentration exceeds 80% by volume, there may be a problem in that the extraction efficiency is not high compared to the solvent concentration. It is preferable to use an ethanol aqueous solution of
상기 딸기 꼭지 열수 추출물 및/또는 알코올 추출물 제조공정에 있어서, 상기 3단계는 1 ~ 3회 반복 수행할 수 있다. 그리고, 4단계의 진공감압농축은 당업계에서 사용하는 일반적인 진공감압농축법으로 수행할 수 있으며, 그 방법을 특별하게 한정하지 않는다. 또한, 상기 동결건조 역시 당업계에서 사용하는 일반적인 동결건조법으로 수행할 수 있다.In the manufacturing process of the strawberry stem hot water extract and/or alcohol extract, the above three steps may be repeated 1 to 3 times. In addition, the vacuum concentration in step 4 may be performed by a general vacuum concentration method used in the art, and the method is not particularly limited. In addition, the freeze-drying may also be performed by a general freeze-drying method used in the art.
이러한 방법으로 제조된 본 발명의 딸기 꼭지 열수 추출물 및/또는 알코올 추출물은 항산화 활성 효과가 우수하며, 항염증 효과가 우수하다.The strawberry stem hot water extract and/or alcohol extract of the present invention prepared in this way has excellent antioxidant activity and anti-inflammatory effect.
본 발명의 딸기 꼭지 추출물은 총 폴리페놀 함량이 1.80 ~ 4.50 mg tanninc acid/g일 수 있으며, 바람직하게는 2.00 ~ 4.50 mg tanninc acid/g, 더욱 바람직하게는 2.30 ~ 4.00 mg tanninc acid/g일 수 있다.The strawberry stem extract of the present invention may have a total polyphenol content of 1.80 to 4.50 mg tanninc acid/g, preferably 2.00 to 4.50 mg tanninc acid/g, and more preferably 2.30 to 4.00 mg tanninc acid/g. there is.
또한, 본 발명의 딸기 꼭지 추출물은 DPPH 라디칼 소거능 측정시, 추출물의 농도가 100 ppm일 때, 하기 식 1에 의거한 DPPH 라디칼 소거능은 40% 이상, 바람직하게는 80.00% 이상, 더욱 바람직하게는 80.0 ~ 88.00%일 수 있다.In addition, when measuring the DPPH radical scavenging activity of the strawberry stem extract of the present invention, when the concentration of the extract is 100 ppm, the DPPH radical scavenging activity based on the following formula 1 is 40% or more, preferably 80.00% or more, more preferably 80.0 ~ 88.00%.
[식 1][Equation 1]
DPPH 라디칼 소거능(%) = {1-(샘플 흡광도/음성대조군 흡광도)}×100%DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
식 1의 샘플 흡광도는 딸기 꼭지 추출물 0.5 ml에 DPPH를 60 μM 로 포함하는 에탄올 용액 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정한 것이고, 음성대조군 흡광도는 증류수 0.5 ml에 DPPH를 60 μM 로 포함하는 에탄올 용액 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정한 것이다.The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 0.5 ml of strawberry stem extract with 3 ml of an ethanol solution containing 60 μM DPPH and reacting in the dark for 15 minutes. 3 ml of an ethanol solution containing 60 μM DPPH was mixed with 0.5 ml, reacted for 15 minutes in a dark room, and then measured at 517 nm using a spectrophotometer.
또한, 본 발명의 딸기 꼭지 추출물은 ABTS 라디칼 소거능 측정시, 추출물의 농도가 100 ppm일 때, 하기 식 2에 의거한 ABTS 라디칼 소거능은 50% 이상, 바람직하게는 90% 이상, 더욱 바람직하게는 95 ~ 99.99%일 수 있다.In addition, when measuring the ABTS radical scavenging activity of the strawberry stem extract of the present invention, when the concentration of the extract is 100 ppm, the ABTS radical scavenging activity based on the following
[식 2][Equation 2]
ABTS 라디칼 소거능(%) = {1-(샘플 흡광도/음성대조군 흡광도)}×100%ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
식 2의 샘플 흡광도는 딸기 꼭지 추출물 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)하 후 3분 간 25℃에서 반응시키고 734 nm에서 흡광도를 측정한 것이고, 음성대조군 흡광도는 증류수 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)하 후 3분 간 25℃에서 반응시키고 734 nm에서 흡광도를 측정한 것이다.The absorbance of the sample in
또한, 본 발명의 딸기 꼭지 추출물은 NO 저해능 측정시, 추출물 농도가 500 ppm(μg/ml)일 때, 하기 식 3에 의거하여 측정한 NO 저해능이 40% 이상, 바람직하게는 일 수 있으며, 바람직하게는 42.0 ~ 55.0%일 수 있다.In addition, the strawberry stem extract of the present invention may have NO inhibition of 40% or more, preferably 40% or more, when the concentration of the extract is 500 ppm (μg/ml), measured according to the following formula 3, when NO inhibition is measured. Preferably, it may be 42.0 to 55.0%.
[식 3][Equation 3]
NO 저해능(%) = 대조군의 NO 측정 레벨(%)- 추출물의 NO 측정 레벨(%)NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)
상기 식 3의 NO 저해능은 RAW 264.7 세포에 LPS(Lipopolysaccharide)를 10 μg/ml 처리한 다음 2시간 뒤에 딸기 꼭지 추출물을 일정 농도로 처리한 후, 18시간 동안 배양한 후, 배양액 내의 NO양을 측정한 것이며, 상기 대조군의 NO 측정 레벨은 100%이며, 추출물의 NO 측정 레벨은 측정된 NO양을 대조군 기준으로 %로 환산시킨 값이다.The NO inhibition ability of Equation 3 was measured by treating RAW 264.7 cells with 10 μg/ml of LPS (Lipopolysaccharide), then treating strawberry stem extracts at a
또한, 본 발명의 딸기 꼭지 추출물은 추출물 농도가 500 ppm일 때, 하기 식 4에 의거하여 측정한 iNOS 발현 저해능이 60% 이상일 수 있으며, 바람직하게는 65.0 ~ 80.0%, 더욱 바람직하게는 70.0 ~ 80.0%일 수 있다.In addition, when the concentration of the strawberry stem extract of the present invention is 500 ppm, the iNOS expression inhibition ability measured according to the following formula 4 may be 60% or more, preferably 65.0 to 80.0%, more preferably 70.0 to 80.0 may be %.
[식 4][Equation 4]
iNOS 발현 저해능(%) = 음성대조군의 iNOS 발현율(%)- 추출물의 iNOS 발현율(%)iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)
상기 식 4에서 음성대조군은 자극제인 LPS를 단독으로 처리한 뒤 측정한 iNOS 발현율이며, 추출물의 iNOS 발현율은 딸기꼭지 추출물로 처리한 뒤 측정한 iNOS 발현율이며, 발현율은 측정된 iNOS 발현량을 음성대조군 기준으로 %로 환산시킨 값이다.In Equation 4, the negative control group is the iNOS expression rate measured after treatment with the stimulant LPS alone, the iNOS expression rate of the extract is the iNOS expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the negative control group It is a value converted to % as a standard.
또한, 본 발명의 딸기 꼭지 추출물은 추출물 농도가 500 ppm일 때, 하기 식 5에 의거하여 측정한 COX-2 발현 저해능이 15.0% 이상일 수 있으며, 바람직하게는 17.0 ~ 30.0%, 더욱 바람직하게는 19.0 ~ 25.0%일 수 있다.In addition, when the concentration of the strawberry stem extract of the present invention is 500 ppm, the COX-2 expression inhibitory ability measured according to the following
[식 5][Equation 5]
COX-2 발현 저해능(%) = 음성대조군의 COX-2 발현율(%)- 추출물의 COX-2 발현율(%)COX-2 expression inhibition ability (%) = COX-2 expression rate of negative control group (%) - COX-2 expression rate of extract (%)
상기 식 5에서 음성대조군은 자극제인 LPS를 단독으로 처리한 뒤 측정한 COX-2 발현율이며, 추출물의 iNOS 발현율은 딸기꼭지 추출물로 처리한 뒤 측정한 COX-2 발현율이며, 발현율은 측정된 COX-2 발현량을 음성대조군 기준으로 %로 환산시킨 값이다.In
본 발명의 딸기 꼭지 추출물을 이용하여 과자, 음료수, 사탕, 캡슐, 영양제,화장료 등 다양한 원료로 가공하여 건강기능식품 및/또는 이너뷰티 조성물을 제공할 수 있다.Using the strawberry stem extract of the present invention, it is possible to provide health functional foods and/or inner beauty compositions by processing various raw materials such as confectionery, beverages, candies, capsules, nutrients, and cosmetics.
이상에서 본 발명에 대하여 구현예를 중심으로 설명하였으나 이는 단지 예시일 뿐 본 발명의 구현예를 한정하는 것이 아니며, 본 발명의 실시예가 속하는 분야의 통상의 지식을 가진 자라면 본 발명의 본질적인 특성을 벗어나지 않는 범위에서 이상에 예시되지 않은 여러 가지의 변형과 응용이 가능함을 알 수 있을 것이다. 예를 들어, 본 발명의 구현예에 구체적으로 나타난 각 구성 요소는 변형하여 실시할 수 있는 것이다. 그리고 이러한 변형과 응용에 관계된 차이점들은 첨부된 청구 범위에서 규정하는 본 발명의 범위에 포함되는 것으로 해석되어야 할 것이다.In the above, the present invention has been described with a focus on embodiments, but this is only an example and does not limit the embodiments of the present invention, and those skilled in the art to which the embodiments of the present invention belong will appreciate the essential characteristics of the present invention. It will be appreciated that various modifications and applications not exemplified above are possible within a range that does not deviate. For example, each component specifically shown in the embodiment of the present invention can be modified and implemented. And differences related to these modifications and applications should be construed as being included in the scope of the present invention as defined in the appended claims.
[실시예][Example]
실시예 1 : 딸기 꼭지 열수 추출물의 제조Example 1: Preparation of Strawberry Stem Hot Water Extract
딸기 과육으로부터 분리된 딸기 꼭지(꽃받침)을 수득한 후, 이를 세척 및 열풍건조한 다음 파쇄하여 딸기 꼭지 분말을 제조하였다.After obtaining a strawberry stem (calyx) separated from strawberry pulp, it was washed and dried with hot air, and then crushed to prepare strawberry stem powder.
다음으로, 상기 딸기 꼭지 분말을 증류수에 첨가하였다. 이때, 딸기 꼭지 분말과 증류수의 혼합비는 1 : 10 중량비였다.Next, the strawberry stem powder was added to distilled water. At this time, the mixing ratio of strawberry stalk powder and distilled water was 1:10 by weight.
다음으로, 100℃에서 3시간 환류 냉각 추출하여 추출액을 수득한 다음, 이를25℃에서 24시간 동안 침지한 후 부직포로 1차 여과하여 상등액과 침전물을 분리하였으며, 상기 여과 및 분리를 3회 반복하였다.Next, an extract was obtained by reflux cooling extraction at 100 ° C. for 3 hours, then immersed at 25 ° C. for 24 hours, and then filtered first with a non-woven fabric to separate the supernatant and the precipitate, and the filtration and separation were repeated three times. .
이때, 여과 및 분리는 진공펌프와 여과지(Whatman No .5, No. 4, No .2)를 이용하여 여과하여 수행하였다.At this time, filtration and separation were performed by filtration using a vacuum pump and filter paper (Whatman No.5, No. 4, No.2).
다음으로, 침전물로부터 분리한 상등액을 로터리 진공 증류기(ratory vacuum진공감evaporator, EYEKA, Japan)을 사용하여 감압농축을 수행하여 농축물을 수득한 후, -20 ℃에서 동결건조 및 보관하여 딸기 꼭지 열수 추출물을 수득하였다.Next, the supernatant separated from the precipitate was concentrated under reduced pressure using a rotary vacuum evaporator (EYEKA, Japan) to obtain a concentrate, and then freeze-dried and stored at -20 ° C to obtain strawberry stem hot water. An extract was obtained.
실시예 2 : 딸기 꼭지 에탄올 추출물의 제조Example 2: Preparation of Strawberry Stem Ethanol Extract
딸기 과육으로부터 분리된 딸기 꼭지(꽃받침)을 수득한 후, 이를 세척 및 열풍건조한 다음 파쇄하여 딸기 꼭지 분말을 제조하였다.After obtaining a strawberry stem (calyx) separated from strawberry pulp, it was washed and dried with hot air, and then crushed to prepare strawberry stem powder.
다음으로, 상기 딸기 꼭지 분말을 70부피% 농도의 에탄올 수용액에 첨가하였다. 이때, 딸기 꼭지 분말과 에탄올 수용액의 혼합비는 1 : 10 중량비였다.Next, the strawberry stalk powder was added to an aqueous ethanol solution having a concentration of 70% by volume. At this time, the mixing ratio of strawberry stalk powder and aqueous ethanol solution was 1:10 by weight.
다음으로, 25℃에서 24시간 동안 교반시켜준 후 부직포로 1차 여과하여 상등액과 침전물을 분리하였으며, 상기 여과 및 분리를 3회 반복하였다.Next, after stirring at 25 ° C. for 24 hours, the supernatant and the precipitate were separated by primary filtration with a non-woven fabric, and the filtration and separation were repeated three times.
이때, 여과 및 분리는 진공펌프와 여과지(Whatman No .5, No. 4, No .2)를 이용하여 여과하여 수행하였다.At this time, filtration and separation were performed by filtration using a vacuum pump and filter paper (Whatman No.5, No. 4, No.2).
다음으로, 침전물로부터 분리한 상등액을 로터리 진공 증류기(ratory vacuum진공감evaporator, EYEKA, Japan)을 사용하여 감압농축을 수행하여 농축물을 수득한 후, -20 ℃에서 동결건조 및 보관하여 딸기 꼭지 에탄올 추출물을 수득하였다.Next, the supernatant separated from the precipitate was concentrated under reduced pressure using a rotary vacuum evaporator (EYEKA, Japan) to obtain a concentrate, and then freeze-dried and stored at -20 ° C to obtain strawberry stem ethanol. An extract was obtained.
실험예 1 : 총 폴리페놀 함량 측정Experimental Example 1: Total polyphenol content measurement
상기 실시예 1의 딸기 꼭지 열수 추출물 및 실시예 2의 딸기 꼭지 알코올 추출물의 총 폴리페놀 함량을 Folin-Denis법을 하기와 같이 변형하여 측정하였고, 그 결과를 하기 표 1에 나타내었다.The total polyphenol content of the strawberry stem hot water extract of Example 1 and the strawberry stem alcohol extract of Example 2 was measured by modifying the Folin-Denis method as follows, and the results are shown in Table 1 below.
측정 방법은 추출물을 증류수에 희석한 다음 2ml과 50% Folin-ciocalteu reagent 2 ml를 혼합한 후 실온에 3분간 방치하였다. 그 후 10% Ca2CO3 2 ml를 가하여 혼합 후 상온에서 1시간 동안 방치하였다가 흡광도 700 nm에서 측정하였다.The measurement method was to dilute the extract in distilled water, mix 2ml and 50% Folin-ciocalteu reagent, and then leave it at room temperature for 3 minutes. Thereafter, 2 ml of 10% Ca 2 CO 3 was added, mixed, and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 700 nm.
(딸기꼭지 열수 추출물)Example 1
(Strawberry stem hot water extract)
(딸기꼭지 에탄올 추출물)Example 2
(Strawberry Stem Ethanol Extract)
(mg tannic acid/g)Total polyphenolic content
(mg tannic acid/g)
일반적으로 페놀성 성분들의 함량 수치와 자유라디칼과 높은 상관관계를 가지고 있으며, 페놀 함량이 증가할수록 항산화 등 생리활성 효과에 매우 좋은 효과를 나타내며, 각 시료의 폴리페놀 함량을 측정한 후 시료 g당 함유하고 있는 양을 나타내었다.In general, there is a high correlation between the content of phenolic components and free radicals, and as the phenol content increases, it shows a very good effect on physiological activities such as antioxidant. After measuring the polyphenol content of each sample, the content per gram of sample indicated the amount of
상기 표 1에서 보는 바와 같이 딸기꼭지 열수 추출물 및 에탄올 추출물은 각각 1.98±0.07 mg/g, 2.66±0.85 mg/g의 높은 폴리페놀 함량을 나타내었다.As shown in Table 1, the strawberry stem hot water extract and the ethanol extract showed high polyphenol contents of 1.98±0.07 mg/g and 2.66±0.85 mg/g, respectively.
실험예 2 : 항산화 효과 측정Experimental Example 2: Measurement of antioxidant effect
상기 실시예 1의 딸기 꼭지 열수 추출물 및 실시예 2의 딸기 꼭지 알코올 추출물의 항산화 효과 측정으로서, 전자공여능 및 ABTS 라디칼 소거능 측정을 하기와 같이 수행하였다.As the antioxidant effects of the strawberry stem hot water extract of Example 1 and the strawberry stem alcohol extract of Example 2, electron donating ability and ABTS radical scavenging activity were measured as follows.
(1) 전자공여능 측정(1) Measurement of electron donating ability
전자공여능으로서 DPPH (1,1-diphenyl-2-picrylhydrazyl) 라디칼 소거 활성 실험은 Blois MS의 방법을 변형하여 시행하였으며, 딸기 꼭지 추출물(시료) 0.5 ml에 60 μM DPPH (in EtOH) 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정하였다. 그리고, DPPH 소거능은 하기 식 1에 의거하여 계산하였으며, 그 결과를 하기 표 1 및 도 1에 나타내었다.DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity test as an electron donating ability was conducted by modifying the Blois MS method, and 0.5 ml of strawberry stem extract (sample) was mixed with 3 ml of 60 μM DPPH (in EtOH). and reacted for 15 minutes in a dark room, and then measured at 517 nm using a spectrophotometer. In addition, the DPPH scavenging ability was calculated based on Equation 1 below, and the results are shown in Table 1 and FIG. 1 below.
이때, 음성대조군은 증류수를 사용하였고, 양성대조군으로는 아스코르빈산(Vit.C)를 사용하였다.At this time, distilled water was used as the negative control group, and ascorbic acid (Vit.C) was used as the positive control group.
[식 1][Equation 1]
DPPH 라디칼 소거능(%) = {1-(샘플 흡광도/음성대조군 흡광도)}×100%DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
식 1의 샘플 흡광도는 딸기 꼭지 추출물 0.5 ml에 DPPH를 60 μM 로 포함하는 에탄올 용액 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정한 것이고, 음성대조군 흡광도는 증류수 0.5 ml에 DPPH를 60 μM 로 포함하는 에탄올 용액 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정한 것이다.The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 0.5 ml of strawberry stem extract with 3 ml of an ethanol solution containing 60 μM DPPH and reacting in the dark for 15 minutes. 3 ml of an ethanol solution containing 60 μM DPPH was mixed with 0.5 ml, reacted for 15 minutes in a dark room, and then measured at 517 nm using a spectrophotometer.
표 2의 측정 결과를 살펴보면, DPPH 라디칼 소거능이 실시예 1은 500ppm 이상부터 80% 이상을 보였고, 실시예 2는 100 ppm 이상부터 80% 이상의 높은 라디칼 소거능을 보였다. 이를 통해서, 실시예 1 ~ 2 모두 우수한 DPPH 라디칼 소거능을 가지지만, DPPH 라디칼 소거능 측면에서 열수 추출물 보다 에탄올 추출물이 상대적으로 우수함을 확인할 수 있었다.Looking at the measurement results of Table 2, the DPPH radical scavenging activity of Example 1 was 80% or more from 500 ppm or more, and Example 2 showed a high radical scavenging activity of 80% or more from 100 ppm or more. Through this, although all of Examples 1 and 2 had excellent DPPH radical scavenging activity, it was confirmed that the ethanol extract was relatively superior to the hot water extract in terms of DPPH radical scavenging activity.
(2) ABTS 라디칼 소거능 측정(2) Measurement of ABTS radical scavenging ability
ABTS 라디칼 소거능은 Nicoletta의 방법3)을 따라 측정하였다. 7 mM ABTS 5 ml와 140 mM, K2S2O8 88 μl를 섞어 암실에 14 ~ 16시간 반응시켰다. 이를 에탄올(absolute ethanol)과 1:88 비율로 섞어 734 nm에서 흡광도 값이 0.7±0.002가 되도록 조절한 ABTS 용액을 제조하였다.ABTS radical scavenging ability was measured according to Nicoletta's method 3). 5 ml of 7 mM ABTS and 88 μl of 140 mM K 2 S 2 O 8 were mixed and reacted in the dark for 14 to 16 hours. This was mixed with ethanol (absolute ethanol) in a ratio of 1:88 to prepare an ABTS solution having an absorbance value of 0.7±0.002 at 734 nm.
딸기 꼭지 추출물(시료) 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)한 후 2.3분 간 상온에서 반응시키고 734 nm에서 흡광도를 측정하였다. 그리고, ABTS 라디칼 소거능은 하기 식 2에 의거하여 계산하였으며, 그 결과를 하기 표 2 및 도 2에 나타내었다.150 μl of strawberry stem extract (sample) and 3 ml of ABTS solution were mixed, vortexed for 30 seconds, reacted at room temperature for 2.3 minutes, and absorbance was measured at 734 nm. In addition, the ABTS radical scavenging ability was calculated based on
이때, 음성대조군은 증류수를 사용하였고, 양성대조군으로는 아스코르빈산(Vit.C)를 사용하였다.At this time, distilled water was used as the negative control group, and ascorbic acid (Vit.C) was used as the positive control group.
[식 2][Equation 2]
ABTS 라디칼 소거능(%) = {1-(샘플 흡광도/음성대조군 흡광도)}×100%ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
식 2의 샘플 흡광도는 딸기 꼭지 추출물 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)하 후 3분 간 25℃에서 반응시키고 734 nm에서 흡광도를 측정한 것이고, 음성대조군 흡광도는 증류수 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)하 후 3분 간 25℃에서 반응시키고 734 nm에서 흡광도를 측정한 것이다.The absorbance of the sample in
표 3의 측정 결과를 살펴보면, ABTS 라디칼 소거능이 실시예 1은 100ppm 이상부터 50% 이상을 보였고, 실시예 2는 50 ppm 이상부터 50% 이상의 높은 라디칼 소거능을 보였으며, 실시예 2는 100 ppm에서 99.0% 이상의 높은 소거능을 보였다. 그리고, 500 μg/ml 및 1,000 μg/ml 농도에서 열수 및 에탄올 추출물은 양성대조군인 Vitamin C와 유사한 활성이 나타났다.Looking at the measurement results of Table 3, Example 1 showed an ABTS radical scavenging ability of 50% or more from 100 ppm or more, Example 2 showed a high radical scavenging ability of 50% or more from 50 ppm or more, and Example 2 showed a high radical scavenging ability of 50% or more from 100 ppm or more. It showed a high scavenging ability of 99.0% or more. In addition, at concentrations of 500 μg/ml and 1,000 μg/ml, the hot water and ethanol extracts showed similar activities as the positive control group, Vitamin C.
이를 통해서, 실시예 1 ~ 2 모두 우수한 ABTS 라디칼 소거능을 가지지만, ABTS 라디칼 소거능 측면에서 열수 추출물 보다 에탄올 추출물이 상대적으로 우수함을 확인할 수 있었다.Through this, although all of Examples 1 and 2 had excellent ABTS radical scavenging activity, it was confirmed that the ethanol extract was relatively superior to the hot water extract in terms of ABTS radical scavenging activity.
실험예 3 : 세포 독성 측정Experimental Example 3: Measurement of cytotoxicity
딸기 꼭지 추출물이 세포에 노출되었을 때 세포에 미치는 독성을 확인하기 위하여 MTT assay를 진행하였다.MTT assay was performed to confirm the toxicity of strawberry stem extract to cells when exposed to cells.
먼저 96 웰 세포배양기에 1×10⁴ cells/well 되도록 RAW264.7 세포를 분주한 후 24시간 뒤에 각 농도에 맞도록 DMSO(dimethyl sulfoxide)에 녹인 샘플을 첨가하였다. 24시간 뒤에 배지를 제거한 후 MTT(2-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) 시약 (in PBS 2.5 mg/ml) well에 40 μl씩 가하고 세포배양기(incubator)에 4시간 반응 후 다시 상층액을 제거하였다.First, after dispensing RAW264.7 cells to 1 × 10⁴ cells/well in a 96-well cell culture medium, samples dissolved in DMSO (dimethyl sulfoxide) were added to suit each concentration after 24 hours. After removing the medium after 24 hours, 40 μl of MTT (2-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) reagent (in PBS 2.5 mg/ml) was added to the well, and the cell culture medium ( After reacting for 4 hours in the incubator), the supernatant was removed again.
다음으로, DMSO를 100 μl씩 분주하여 완전히 녹인 후, 분광광도계를 이용하여 흡광도 540 nm에서 측정하였고, 그 결과를 도 3에 나타내었다.Next, after dispensing 100 μl of DMSO and completely dissolving it, the absorbance was measured at 540 nm using a spectrophotometer, and the results are shown in FIG. 3 .
RAW264.7의 세포 독성 측정 결과, 실시예 1(딸기꼭지 열수 추출물) 및 실시예 2(에탄올 추출물)은 500 μg/ml 농도에서 90% 이상의 높은 세포 생존율이 나타나는 것을 확인하였다.As a result of measuring the cytotoxicity of RAW264.7, it was confirmed that Example 1 (hot water extract of strawberry stem) and Example 2 (ethanol extract) showed high cell viability of 90% or more at a concentration of 500 μg/ml.
이러한 결과를 바탕으로, 세포 생존율이 90% 이상인 500 μg/ml 농도인 추출물을 사용하여, 이하에서 NO, iNOS 및 COX-2의 단백질 발현 측정 시험을 진행하였다.Based on these results, using an extract having a concentration of 500 μg/ml having a cell viability of 90% or more, NO, iNOS, and COX-2 protein expression measurement tests were performed below.
실험예 4 : 항염증 측정Experimental Example 4: Anti-inflammatory measurement
(1) 일산화질소(NO) 저해능 측정(1) Measurement of nitric oxide (NO) inhibition
염증반응의 대표적인 인자인 NO의 생성 억제하는지 확인하기 위해 RAW 264.7 세포에 LPS(Lipopolysaccharide)를 10 μg/ml 처리한 다음 2시간 뒤에 추출물을 각 농도별로 처리하였다. 18시간 동안 배양 후 배양액 내의 NO양을 측정하였고, 그 결과를 도 4의 A, B에 나타내었다.In order to determine whether production of NO, a representative factor of the inflammatory response, was inhibited, RAW 264.7 cells were treated with 10 μg/ml of LPS (Lipopolysaccharide), and then the extract was treated at each concentration after 2 hours. After culturing for 18 hours, the amount of NO in the culture medium was measured, and the results are shown in A and B of FIG. 4 .
NO 저해 활성 측정은, RAW 264.7 세포의 상청액(supernatant)에서의 NO 양을 아질산염(nitrite)과 질산염(nitrate)로서 측정하였다. 아질산염에 대한 질산염으로 환원된 후의 안전한 형태인 그리스 시약(griess reagent)을 사용하였으며, 96웰 세포배양기 1×10⁴cells/well로 분주된 RAW264.7 세포의 컨플루언스(confluence)가 80%일 때, PBS(phosphate buffered saline)로 2번 수세(washing)한 후에 무혈청 배지를 사용하여 18시간 이상 배양시킨 후에 시료를 농도별로 처리하여 1시간 동안 배양한 후 LPS(lipopolysaccharide) 1 μg/ml 처리하고 24 시간 동안 배양하였다. 배양액의 상층액을 취하여 그리스 시약과 반응시킨 후 ELISA 리더기(reader)로 540 nm에서 흡광도를 측정하여 NO 생성율을 백분율로 표시하였다.NO inhibitory activity measurement, the amount of NO in the supernatant (supernatant) of RAW 264.7 cells was measured as nitrite (nitrite) and nitrate (nitrate). A grease reagent, which is a safe form after reduction with nitrate for nitrite, was used, and when the confluence of RAW264.7 cells dispensed at 1 × 10⁴cells/well in a 96-well cell culture medium was 80%, After washing twice with PBS (phosphate buffered saline) and culturing for more than 18 hours using a serum-free medium, the samples were treated by concentration and cultured for 1 hour, treated with 1 μg/ml of LPS (lipopolysaccharide), and then cultured for 24 hours. incubated for hours. The supernatant of the culture was reacted with a grease reagent, and then the absorbance was measured at 540 nm using an ELISA reader, and the NO production rate was expressed as a percentage.
LPS 단독 처리군과 LPS 무처리군에서의 차이가 나타나는 것을 확인하였으며, LPS와 농도별로 추출물을 처리하였을 때, 실시예 1(딸기꼭지 열수 추출물) 및 실시예 2(딸기꼭지 에탄올 추출물)은 농도 의존적으로 NO 생성량이 감소하는 것을 확인하였다. 이때, NO 저해능은 하기 식 3에 의거하여 측정하였다.It was confirmed that there was a difference between the LPS-only treatment group and the LPS-untreated group, and when the extracts were treated by LPS and concentration, Example 1 (strawberry stem hot water extract) and Example 2 (strawberry stem ethanol extract) showed concentration-dependent It was confirmed that NO production decreased. At this time, the NO inhibition ability was measured based on Equation 3 below.
500 μg/ml 농도에서 실시예 1(딸기꼭지 열수 추출물)은 49.45%, 실시예 2(딸기꼭지 에탄올 추출물)은 44.48%의 저해 효과를 확인하였다.At a concentration of 500 μg/ml, Example 1 (hot water extract of strawberry stems) had an inhibitory effect of 49.45% and Example 2 (ethanol extract of strawberry stems) had an inhibitory effect of 44.48%.
[식 3][Equation 3]
NO 저해능(%) = 대조군의 NO 측정 레벨(%)- 추출물의 NO 측정 레벨(%)NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)
식 3의 NO 저해능은 RAW 264.7 세포에 LPS를 10 μg/ml 처리한 다음 2시간 뒤에 딸기 꼭지 추출물을 일정 농도로 처리한 후, 18시간 동안 배양한 후, 배양액 내의 NO양을 측정한 것이며, 상기 대조군의 NO 측정 레벨은 100%이며, 추출물의 NO 측정 레벨은 측정된 NO양을 대조군 기준으로 %로 환산시킨 값이다.The NO inhibition ability of Formula 3 was obtained by treating RAW 264.7 cells with 10 μg/ml of LPS, then treating strawberry stem extract at a
(2) 웨스턴 블랏(western blot)을 통한 iNOS, COX-2발현 저해능 측정(2) Measurement of iNOS and COX-2 expression inhibition by western blot
염증의 매개 인자인 iNOS, COX-2의 단백질 발현량에 미치는 실시예 1(딸기꼭지 열수 추출물) 및 실시예 2(딸기꼭지 에탄올 추출물)의 항염증 효과를 확인하였고, 그 결과를 도 5의 A와 B(iNOS 발현) 및 도 6의 A와 B(COX-2 발현)에 나타내었다.The anti-inflammatory effects of Example 1 (hot water extract of strawberry stem) and Example 2 (ethanol extract of strawberry stem) on the protein expression levels of iNOS and COX-2, which are mediators of inflammation, were confirmed, and the results are shown in FIG. and B (iNOS expression) and FIG. 6 A and B (COX-2 expression).
단백질 발현량 측정 방법은 RAW264.7 세포를 10% FBS(fetal bovine serum)를 포함한 DMEM(Dulbecco's modified Eagle's medium)에 현탁시킨 후, 6 웰 세포배양기에 2×105 cells/well의 세포수가 되도록 3 ml씩 분주하여 37℃, 5% CO2 배양기에서 24시간 배양하였다. 다음으로, 새로운 DMEM 배지로 교환한 후 2시간 동안 자극제인 LPS를 처리하여 배양한 후 딸기꼭지 추출물을 농도별로 세포에 처리하여 배양하였다. 24시간 후 단백질 발현을 측정하기 위하여 배지를 제거하고 차가운 PBS로 세척한 후 세포 용해물(cell lysates)는 RIPA 버퍼(buffer)에 Protease&Phosphatase Single-Use inhibitor Cocktail 100X를 첨가하여 단백질을 추출하였다. 단백질을 정량(BCA protein kit)하여, 10% SDS-PAGE에 전기영동한 후, 멤브레인(membrane)으로 이주(transfer)하여 5% 탈지유(5% skim milk in TBST)에 2시간 동안 블로킹(blocking)하였다. 1차 항체를 희석(3% skim milk in TBST)하여 4℃에서 오버나이트(overnight)한 다음, TBST(Tris buffered saline with tween 20)로 3회 세척하였다. 2차 항체는 실온에서 2시간 동안 배양하여 측정하였다.To measure the protein expression level, RAW264.7 cells were suspended in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (fetal bovine serum), and then cultured in a 6-well cell culture medium so that the cell number was 2×10 5 cells/well. Divided by ml, and cultured for 24 hours in a 37°C, 5% CO 2 incubator. Next, the cells were cultured by treating the stimulant, LPS, for 2 hours after exchanging with a new DMEM medium, and then treating the cells with strawberry stem extract at each concentration and cultured. After 24 hours, to measure protein expression, the medium was removed, washed with cold PBS, and protein was extracted from the cell lysates by adding Protease & Phosphatase Single-Use inhibitor Cocktail 100X to RIPA buffer. Protein was quantified (BCA protein kit), electrophoresed on 10% SDS-PAGE, transferred to a membrane, and blocked in 5% skim milk in TBST for 2 hours did The primary antibody was diluted (3% skim milk in TBST), overnight at 4°C, and washed three times with TBST (Tris buffered saline with tween 20). Secondary antibodies were measured by incubation at room temperature for 2 hours.
iNOS 및 COX-2의 단백질 발현은 ECL(enhanced chemiluminescence) 용액(Amersham, Pittsburgh, PA, USA)을 이용하여 확인하였다. 내부통제9Internal control)로써 1:1000으로 희석한 베타-엑틴(β-actin, Santa Cruz Biotechnology, Santa Cruz, California, USA)을 사용하였다.The protein expression of iNOS and COX-2 was confirmed using an enhanced chemiluminescence (ECL) solution (Amersham, Pittsburgh, PA, USA). 1:1000 diluted beta-actin (Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as an internal control (9).
음성대조군은(Con) 딸기꼭지 추출물 대신 자극제인 LPS 단독으로 처리한 것이고, 정상군(Nor)은 자극제인 LPS와 딸기꼭지 추출물을 처리하지 않은 것이다.The negative control group (Con) was treated with the stimulant LPS alone instead of the strawberry stem extract, and the normal group (Nor) was not treated with the stimulant LPS and strawberry stem extract.
그리고, iNOS 발현 저해능은 하기 식 4에 의거하여 계산하였고, COX-2 발현 저해능은 식 5에 의거하여 계산하였다.In addition, the ability to inhibit iNOS expression was calculated based on Equation 4 below, and the ability to inhibit COX-2 expression was calculated based on
[식 4][Equation 4]
iNOS 발현 저해능(%) = 음성대조군의 iNOS 발현율(%)- 추출물의 iNOS 발현율(%)iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)
상기 식 4의 iNOS 발현 저해능은 iNOS 발현량을 측정한 것이며, 상기 음성대조군의 iNOS 발현율은 100%이며, 추출물의 iNOS 발현율은 측정된 iNOS 발현량을 대조군 기준으로 %로 환산시킨 값이다.The ability to inhibit iNOS expression in Equation 4 is obtained by measuring the iNOS expression level, the iNOS expression level of the negative control group is 100%, and the iNOS expression level of the extract is the value obtained by converting the measured iNOS expression level to % based on the control group.
[식 5][Equation 5]
COX-2 발현 저해능(%) = 음성대조군의 COX-2 발현율(%)- 추출물의 COX-2 발현율(%)COX-2 expression inhibition ability (%) = COX-2 expression rate of negative control group (%) - COX-2 expression rate of extract (%)
상기 식 5의 COX-2 발현 저해능은 COX-2 발현량을 측정한 것이며, 상기 대조군의 COX-2 발현율은 100%이며, 추출물의 COX-2 발현율은 측정된 COX-2 발현량을 대조군 기준으로 %로 환산시킨 값이다.The ability to inhibit COX-2 expression in
도 5 및 도 6을 살펴보면, RAW 264.7 세포에 추출물을 10, 50, 100, 500 μg/ml 농도로 처리하고 각 단백질의 발현량을 측정한 결과 딸기꼭지 열수 추출물의 최종 농도 구간인 500 μg/ml 농도에서 iNOS, COX-2의 단백질 발현은 65.79%, 22.99% 저해하는 것을 확인할 수 있었다.5 and 6, RAW 264.7 cells were treated with extracts at concentrations of 10, 50, 100, and 500 μg/ml, and the expression level of each protein was measured. It was confirmed that the protein expression of iNOS and COX-2 was inhibited by 65.79% and 22.99% at each concentration.
또한, 딸기꼭지 70% EtOH 추출물의 최종 농도 구간인 500 μg/ml 농도에서 iNOS, COX-2의 단백질 발현은 73.98%, 19.44% 저해하는 것을 확인할 수 있었다.In addition, it was confirmed that the protein expression of iNOS and COX-2 was inhibited by 73.98% and 19.44% at the final concentration of 500 μg/ml of the strawberry stem 70% EtOH extract.
상기 실시예 및 실험예를 통하여, 본 발명의 딸기꼭지 열수 추출물 및/또는 알코올 추출물이 우수한 항산화 및 항염증 효과가 있는 것을 확인할 수 있었으며, 이러한 본 발명의 딸기꼭지 추출물을 다양한 이너뷰티 소재로 적용하기 적합함을 확인할 수 있었다.Through the above examples and experimental examples, it was confirmed that the hot water extract and/or alcohol extract of the present invention had excellent antioxidant and anti-inflammatory effects, and the strawberry stem extract of the present invention was applied to various inner beauty materials. fit could be confirmed.
본 발명의 단순한 변형이나 변경은 이 분야의 통상의 지식을 가진 자에 의해서 용이하게 실시될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.Simple modifications or changes of the present invention can be easily performed by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.
Claims (9)
상기 딸기 꼭지 분말 및 65 ~ 80 부피% 농도의 에탄올 수용액을 1 : 8 ~ 12 중량비로 혼합한 후, 20 ~ 30℃에서 18 ~ 30시간 동안 교반을 수행하는 2단계;
2단계를 수행한 추출액을 여과하여 상등액과 침전물을 분리하는 3단계;
상등액을 진공감압농축을 수행하여 농축물을 수득한 후, 동결건조를 수행하여 알코올 추출물을 수득하는 4단계;를 포함하는 공정을 수행하며,
상기 3단계는 1 ~ 3회 반복 수행하고,
4단계의 상기 알코올 추출물은 총 폴리페놀 함량이 2.30 ~ 4.50 mg tanninc acid/g이며 ,
상기 딸기 꼭지 추출물은 추출물 농도가 100 ppm일 때, 하기 식 1에 의거하여 측정한 1,1-다이페닐-2-피크릴하이드라질(DPPH, 1,1-diphenyl-2-picrylhydrazyl) 라디칼 소거능이 80.00% 이상이며,
상기 딸기 꼭지 추출물은 추출물 농도가 100 ppm일 때, 하기 식 2에 의거하여 측정한 2,2'-아지노-비스(3-에틸벤조티아졸린-6-설포닉애시드(ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 라디칼 소거능이 90% 이상이고,
상기 딸기 꼭지 추출물은 추출물 농도가 500 ppm일 때, 하기 식 3에 의거하여 측정한 NO 저해능이 40% 이상이며,
상기 딸기 꼭지 추출물은 추출물 농도가 500 ppm일 때, 하기 식 4에 의거하여 측정한 산화질소 합성효소(iNOS, Nitric oxide synthase) 발현 저해능이 60% 이상이고, 하기 식 5에 의거하여 측정한 프로스타글란딘-엔도페록시드 합성효소 2(COX-2, Prostaglandin-endoperoxide synthase 2) 발현 저해능이 15.0% 이상인 것을 특징으로 하는 항산화 및 항염증성 이너뷰티용 딸기 꼭지 추출물의 제조방법.
[식 1]
DPPH 라디칼 소거능(%) = {1-(샘플 흡광도/음성대조군 흡광도)}×100%
식 1의 샘플 흡광도는 딸기 꼭지 추출물 0.5 ml에 DPPH를 60 μM 로 포함하는 에탄올 용액 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정한 것이고, 음성대조군 흡광도는 증류수 0.5 ml에 DPPH를 60 μM 로 포함하는 에탄올 용액 3 ml을 혼합하여 암실에서 15분간 반응한 다음 분광광도계를 사용하여 517 nm에서 측정한 것이다.
[식 2]
ABTS 라디칼 소거능(%) = {1-(샘플 흡광도/음성대조군 흡광도)}×100%
식 2의 샘플 흡광도는 딸기 꼭지 추출물 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)한 후, 3분간 25℃에서 반응시키고, 734 nm에서 흡광도를 측정한 것이고, 음성대조군 흡광도는 증류수 150 μl와 ABTS 용액 3 ml를 혼합하여 30초간 보텍스(vortex)한 후, 3분간 25℃에서 반응시키고, 734 nm에서 흡광도를 측정한 것이다.
[식 3]
NO 저해능(%) = 대조군의 NO 측정 레벨(%)- 추출물의 NO 측정 레벨(%)
식 3의 NO 저해능은 RAW 264.7 세포에 LPS(Lipopolysaccharide)를 10 μg/ml 처리한 다음 2시간 뒤에 딸기 꼭지 추출물을 일정 농도로 처리한 후, 18시간 동안 배양한 후, 배양액 내의 NO양을 측정한 것이며, 상기 대조군의 NO 측정 레벨은 100%이며, 추출물의 NO 측정 레벨은 측정된 NO양을 대조군 기준으로 %로 환산시킨 값이다.
[식 4]
iNOS 발현 저해능(%) = 음성대조군의 iNOS 발현율(%)- 추출물의 iNOS 발현율(%)
상기 식 4에서 음성대조군은 자극제인 LPS를 단독으로 처리한 뒤 측정한 iNOS 발현율이며, 추출물의 iNOS 발현율은 딸기꼭지 추출물로 처리한 뒤 측정한 iNOS 발현율이며, 발현율은 측정된 iNOS 발현량을 음성대조군 기준으로 %로 환산시킨 값이다.
[식 5]
COX-2 발현 저해능(%) = 음성대조군의 COX-2 발현율(%)- 추출물의 COX-2 발현율(%)
상기 식 5에서 음성대조군은 자극제인 LPS를 단독으로 처리한 뒤 측정한 COX-2 발현율이며, 추출물의 iNOS 발현율은 딸기꼭지 추출물로 처리한 뒤 측정한 COX-2 발현율이며, 발현율은 측정된 COX-2 발현량을 음성대조군 기준으로 %로 환산시킨 값이다.Step 1 of preparing strawberry stem powder by washing and drying strawberry stems and pulverizing them;
A second step of mixing the strawberry stem powder and 65 to 80% by volume aqueous ethanol solution at a weight ratio of 1:8 to 12, followed by stirring at 20 to 30 ° C. for 18 to 30 hours;
Step 3 of separating the supernatant and the precipitate by filtering the extract obtained in step 2;
Performing a process comprising: performing a vacuum concentration of the supernatant to obtain a concentrate, followed by lyophilization to obtain an alcohol extract;
The above 3 steps are repeated 1 to 3 times,
The alcohol extract in step 4 has a total polyphenol content of 2.30 to 4.50 mg tanninc acid / g,
The strawberry stem extract has 1,1-diphenyl-2-picrylhydrazyl (DPPH, 1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity measured according to the following formula 1 when the extract concentration is 100 ppm more than 80.00%,
The strawberry stem extract was 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS, 2,2'- The azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging ability is over 90%,
When the concentration of the strawberry stem extract is 500 ppm, the NO inhibition ability measured according to the following formula 3 is 40% or more,
The strawberry stem extract has an inhibitory ability of nitric oxide synthase (iNOS) expression of 60% or more, measured according to the following formula 4, when the extract concentration is 500 ppm, and a prostaglandin-measured according to the following formula 5 A method for producing strawberry stem extract for antioxidant and anti-inflammatory inner beauty, characterized in that the expression inhibition of endoperoxide synthase 2 (COX-2, Prostaglandin-endoperoxide synthase 2) is 15.0% or more.
[Equation 1]
DPPH radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
The absorbance of the sample in Equation 1 was measured at 517 nm using a spectrophotometer after mixing 0.5 ml of strawberry stem extract with 3 ml of an ethanol solution containing 60 μM DPPH and reacting in the dark for 15 minutes. 3 ml of an ethanol solution containing 60 μM DPPH was mixed with 0.5 ml, reacted for 15 minutes in a dark room, and then measured at 517 nm using a spectrophotometer.
[Equation 2]
ABTS radical scavenging ability (%) = {1-(sample absorbance/negative control absorbance)}×100%
The absorbance of the sample in Equation 2 was obtained by mixing 150 μl of strawberry stem extract and 3 ml of ABTS solution, vortexing for 30 seconds, reacting at 25 ° C for 3 minutes, and measuring the absorbance at 734 nm. After mixing 150 μl and 3 ml of ABTS solution, vortexing for 30 seconds, reacting at 25° C. for 3 minutes, and measuring absorbance at 734 nm.
[Equation 3]
NO inhibition ability (%) = measured NO level of control (%) - measured NO level of extract (%)
The NO inhibition ability of Equation 3 was determined by treating RAW 264.7 cells with 10 μg/ml of LPS (Lipopolysaccharide), then treating strawberry stem extracts at a certain concentration 2 hours later, incubating them for 18 hours, and then measuring the amount of NO in the culture medium. The NO measurement level of the control group is 100%, and the NO measurement level of the extract is a value obtained by converting the measured NO amount to % based on the control group.
[Equation 4]
iNOS expression inhibition ability (%) = iNOS expression rate of negative control group (%) - iNOS expression rate of extract (%)
In Equation 4, the negative control group is the iNOS expression rate measured after treatment with the stimulant LPS alone, the iNOS expression rate of the extract is the iNOS expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the negative control group It is a value converted to % as a standard.
[Equation 5]
COX-2 expression inhibition ability (%) = COX-2 expression rate of negative control group (%) - COX-2 expression rate of extract (%)
In Equation 5, the negative control group is the COX-2 expression rate measured after treatment with the stimulant LPS alone, and the iNOS expression rate of the extract is the COX-2 expression rate measured after treatment with the strawberry stalk extract, and the expression rate is the measured COX-2 expression rate. 2 It is a value obtained by converting the expression level to % based on the negative control group.
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