KR100791282B1 - Pimpinella komarovii extracts having whitening activity - Google Patents

Pimpinella komarovii extracts having whitening activity Download PDF

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KR100791282B1
KR100791282B1 KR1020070019697A KR20070019697A KR100791282B1 KR 100791282 B1 KR100791282 B1 KR 100791282B1 KR 1020070019697 A KR1020070019697 A KR 1020070019697A KR 20070019697 A KR20070019697 A KR 20070019697A KR 100791282 B1 KR100791282 B1 KR 100791282B1
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extract
roe deer
pimpinella
komarovii
activity
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김기옥
한종헌
김봉석
강민철
김미량
고광효
이주엽
진호경
엄병헌
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재단법인 제주하이테크산업진흥원
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97Cosmetics 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
    • A61K8/9783Angiosperms [Magnoliophyta]
    • A61K8/9789Magnoliopsida [dicotyledons]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/02Preparations for care of the skin for chemically bleaching or whitening the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH

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  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
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  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Birds (AREA)
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  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Cosmetics (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

An extract of Pimpinella komarovii is provided to secure excellent whitening effect and excellent antioxidative and anti-inflammatory activities. And a composition comprising the same as an effective ingredient is provided. An extract of Pimpinella komarovii with whitening activity is characterized in that it is prepared by hot-wind drying washed Pimpinella komarovii at a temperature of 40 deg.C, crushing the dried Pimpinella komarovii, dipping the crushed material of the Pimpinella komarovii in 70% ethanol with agitating for 2-3 days at room temperature, filtering the leachate using a filter to prepare a crude extract, and fractioning the crude extract using n-hexane, ethylacetate, and butanol in sequence. A composition for whitening, improving and preventing inflammatory diseases or antioxidizing comprises the extract of Pimpinella komarovii as an effective ingredient.

Description

미백활성을 나타내는 노루참나물 추출물{PIMPINELLA KOMAROVII EXTRACTS HAVING WHITENING ACTIVITY}Extract of roe deer sprouts showing whitening activity {PIMPINELLA KOMAROVII EXTRACTS HAVING WHITENING ACTIVITY}

도 1은 본 발명의 노루참나물 추출물 및 그로부터 분리된 카페익산 메틸 에스테르의 제조공정도1 is a manufacturing process of the roe deer extract of the present invention and the caffeic acid methyl ester separated therefrom

도 2는 본 발명의 노루참나물 추출물에 대한 HPLC 분석결과 그래프Figure 2 is a graph of HPLC analysis results for roe deer extract of the present invention

도 3은 본 발명의 노루참나물 에틸아세테이트 분획물에 대한 HPLC 분석결과 그래프Figure 3 is a graph of HPLC analysis results for the roe deer ethyl acetate fraction of the present invention

도 4는 본 발명의 노루참나물 부탄올 분획물에 대한 HPLC 분석결과 그래프Figure 4 is a graph of the HPLC analysis results for the roe deer butanol fraction of the present invention

도 5는 본 발명의 노루참나물 유래 단일물질(카페익산 메틸 에스테르)에 대한 HPLC 분석결과 그래프Figure 5 is a graph of HPLC analysis results for a single substance (caffeic acid methyl ester) derived from roe deer sprouts of the present invention.

도 6은 본 발명의 노루참나물 유래 단일물질(카페익산 메틸 에스테르)에 대한 LC-MS 스펙트럼6 is an LC-MS spectrum of a single substance (caffeic acid methyl ester) derived from roe deer sprout of the present invention.

도 7은 본 발명의 노루참나물 유래 단일물질(카페익산 메틸 에스테르)에 대한 1H-NMR 스펙트럼7 is a 1H-NMR spectrum of a single substance (caffeic acid methyl ester) derived from roe deer sprout of the present invention.

도 8은 본 발명의 노루참나물 유래 단일물질(카페익산 메틸 에스테르)에 대한 13C-NMR 스펙트럼8 is a 13C-NMR spectrum of a single substance (caffeic acid methyl ester) derived from roe deer sprouts of the present invention.

도 9는 본 발명의 노루참나물 유래 단일물질(카페익산 메틸 에스테르)에 대한 H-H COSY 스펙트럼Figure 9 is a H-H COSY spectrum for a single substance (caffeic acid methyl ester) derived from roe deer sprouts of the present invention

도 10은 본 발명의 노루참나물 유래 카페익산 메틸 에스테르 구조10 is a caffeic acid methyl ester structure of roe deer sprouts of the present invention

도 11은 본 발명의 노루참나물 추출물에 대한 Melan-a 세포의 세포독성 실험결과 그래프11 is a graph showing the results of cytotoxicity test of Melan-a cells against roe deer extract of the present invention

도 12는 본 발명의 노루참나물 추출물에 대한 멜라닌 합성억제 효과 결과 그래프12 is a graph showing the results of the melanin synthesis inhibitory to the roe deer extract of the present invention

A : 노루참나물 추출물, B : 노루참나물 추출분획물A: Roe deer yam extract, B: Roe deer yam extract

도 13은 본 발명의 노루참나물 추출물에 대한 Melan-a 세포에서의 세포 티로시나제 억제효과 결과 그래프13 is a graph showing the results of cell tyrosinase inhibitory effect on Melan-a cells to the roe deer extract of the present invention

A : 노루참나물 추출물, B : 노루참나물 추출분획물A: Roe deer yam extract, B: Roe deer yam extract

도 14은 본 발명의 노루참나물 추출물에 대한 RAW293.7 세포에서의 항염효과14 is an anti-inflammatory effect in RAW293.7 cells to the roe deer extract of the present invention

A : 세포 NO 에세이 결과, B : 세포 MTT 에세이 결과A: cell NO assay result, B: cell MTT assay result

본 발명은 미백활성을 나타내는 노루참나물 추출물에 관한 것이다.The present invention relates to a roe deer extract extract showing whitening activity.

사람의 피부색은 표피에 존재하는 색소세포인 멜라닌 세포에서 생성하는 피부멜라닌, 헤모글로빈이나 카로티노이드 등의 색소의 유.무 및 피부의 두께와 반사 도 등에 영향을 받는다.Human skin color is affected by the presence or absence of pigments such as melanin, hemoglobin or carotenoids produced by melanocytes, which are pigment cells present in the epidermis, and the thickness and reflectivity of the skin.

이 중 피부색을 결정하는데 가장 중요한 요소인 멜라닌 색소는 인체에 정상적으로 존재하는 아미노산의 일종인 티로신(Tyrosin)이 멜라닌 세포내에 존재하는 효소인 티로시네이즈(Tyrosinase)에 의해 도파(DOPA)되고, 계속되는 일련의 복잡한 산화과정을 통해 최종적으로 흑갈색의 중합체인 멜라닌을 생성하게 된다.Among these, melanin pigment, which is the most important factor in determining the skin color, is typified by tyrosinase, an amino acid that is normally present in the human body, and is dopa- ted by tyrosinase, an enzyme in melanocytes. The complex oxidation process leads to melanin, a dark brown polymer.

멜라닌 합성은 자외선 노출, 멜라노마(melanoma), 색소과다침착증( hyperpigmentation disease)등의 요인에 의하여 합성이 촉진된다.Melanin synthesis is promoted by factors such as UV exposure, melanoma and hyperpigmentation disease.

또한, 멜라닌 색소의 생합성은 티로시나제(tyrosinase) 효소를 비롯하여 여러 효소들에 의하여 조절되고 있으며, 그중 티로시나제는 티로신(tyrosin)을 기질로 하여 L-도파퀴논(L-dopaquinone)으로 전이되는 초기 생합성과정이후 디하이드록시인돌(dihydroxyindole)의 산화에 작용한다. In addition, the biosynthesis of melanin pigment is controlled by various enzymes, including tyrosinase enzymes, among which tyrosinase is the initial biosynthesis process of tyrosine (Trosin) as a substrate to L-dopaquinone It acts on the oxidation of dihydroxyindoles.

따라서, 티로시나제 활성 억제제를 찾는 연구가 미백제의 개발에 있어서 중요한 부분을 차지하고 있으며, 현재 계속 알려지고 있는 티로시나제 저해제로 하이드로퀴논(hydroquinone), 4-하이드록시아니졸(4-hydroxyanisole), 아스코르빈산 (ascorbic acid) 유도체, 코지산(kojic acid), 아젤라인산(azelaic acid), 코르티코스테로이드(corticosteroid), 레티노이드(retinoids), 알부틴(arbutin), 카테킨(catechin) 등이 있으나, 이들의 안전성과 경제성 등의 문제점으로 사용에 있어서 어려움이 있다. Therefore, the search for tyrosinase activity inhibitors is an important part of the development of the whitening agent, and as the tyrosinase inhibitors which are still known, hydroquinone, 4-hydroxyanisole, ascorbic acid ( ascorbic acid derivatives, kojic acid, azelaic acid, corticosteroids, retinoids, arbutin, catechin, etc. There is a difficulty in using it as a problem.

이에 따라 최근에는 천연식물을 이용한 미백원료, 기능성 식품, 기능성 화장료 등 각 분야에서 인공물질이 아닌 천연물을 이용한 연구가 활발히 진행되어 지고 있다.Accordingly, in recent years, researches using natural products, rather than artificial substances, in whitening raw materials, functional foods, and functional cosmetics using natural plants have been actively conducted.

한국등록특허공보 10-0542751(티로시나제 저해활성을 지닌 곰피 추출물 또는 그로부터 분리한 플로로탄닌류를 함유하는 조성물)에는, 곰피 추출물 또는 그로부터 분리된 플로로탄닌류의 화합물이 멜라닌 생합성 과정에 핵심적으로 작용하는 효소인 티로시나제에 대해 작용하여, 그 효소활성을 효과적으로 저해하므로, 멜라닌 과다생성을 예방하기 위한 미백용 화장료 조성물 또는 식품 등의 갈변 방지용 조성물에 관한 것이 공개되어 있다.In Korean Registered Patent Publication No. 10-0542751 (composition with gompi extract with tyrosinase inhibitory activity or phlotantanins isolated therefrom), gompi extract or a compound of phlorotannins isolated therefrom plays a key role in melanin biosynthesis process It acts on tyrosinase, which is an enzyme, and effectively inhibits its enzymatic activity, and thus, there is disclosed a composition for preventing browning, such as a whitening cosmetic composition or food for preventing melanin overproduction.

또, 한국등록특허공보 10-0602684(망초 추출물을 유효성분으로 함유하는 미백용 화장료조성물)에는, 망초 추출물이 멜라닌 생성 세포에서의 멜라닌 생성을 저해하고 티로시나제 활성을 억제하며, 항산화활성도 함께 나타내는 피부 미백용 화장료조성물에 관한 것이 공개되어 있다.In addition, Korean Patent Publication No. 10-0602684 (a cosmetic composition for whitening containing the forget-me-not extract) as an active ingredient, skin whitening that the forget-me-not extract inhibits melanin production in melanocytes, inhibits tyrosinase activity, and also has antioxidant activity. A cosmetic composition for cosmetics is disclosed.

한편, 노루참나물(Pimpinella komarovii)은 산형과에 속하는 다년생초본으로서 제주도를 비롯하여 우리나라 북부, 함경남도, 동부시베리아 등지에 자생하고 있다.On the other hand, Pimpinella komarovii is a perennial herb belonging to the mountain family family, which grows in Jeju Island, North Korea, Hamgyongnamdo, and East Siberia.

한국등록특허공보 10-0291089(참나물을 이용한 육류 및 생선의 냄새 제거제의 제조방법 및 냄새제거제)에는, 참나물에 디에틸에티르 용매를 이용하여 추출하고 무수 황산나트륨으로 탈수 및 여과하여 농축한 참나물 추출물을 육류 및 생선의 냄새를 제거제로 이용하는 것에 관한 것이 공개되어 있다.Korean Registered Patent Publication No. 10-0291089 (Method for manufacturing meat and fish odor remover using yam and fish odor remover) extracts yam sprouts extracted with diethyl ether solvent, and dehydrated and filtered with anhydrous sodium sulfate. The use of meat and fish odors as a remover is disclosed.

또한, 한국등록특허공보 10-0624664(비듬균 성장억제 및 두피 가려움 개선용 두발 화장료조성물)에는, 과루인, 후박, 참나물을 유기용매로 추출하여 두피 가려 움 및 비듬 개선이 있는 화장료 조성물로 제조하는 것에 관한 것이 공개되어 있다.In addition, Korean Patent Publication No. 10-0624664 (hair cosmetic composition for the control of dandruff growth and scalp itch improvement), extracting fruit juice, thick pepper, and sesame seed with an organic solvent to produce a cosmetic composition with improved scalp itch and dandruff. Is open to the public.

상기의 발명들과 같이 종래에는 참나물(Pimpinella)을 이용한 추출물이 두피 가려움 및 비듬개선용 화장료 조성물이나 육류 및 생선의 냄새제거제로 활용되는 조성물이 공개되어 있으며, 참나물속(Pimpinella) 중 몇몇 종들에서 기관지 천식 및 위장장애 치료제, 구풍제, 진경제, 거담제, 진위제 등등 약재와 향신료, 식용으로 쓰인다고 알려져 있는 정도로써, 현재 노루참나물(Pimpinella komarovii)에 대한 생리활성 연구는 부족한 실정이다.As the above inventions, conventionally, extracts using sesame sprouts ( Pimpinella ) are used as cosmetic compositions for improving scalp itch and dandruff or as deodorants of meat and fish, and bronchial bronchus in some species of Pimpinella The research on physiological activity of Pimpinella komarovii is insufficient, as it is known to be used for medicines, spices, and foods for treating asthma and gastrointestinal disorders, antipyretics, antispasmodics, expectorants, and anti-tussives.

본 발명은 미백 활성이 뛰어나고, 항산화 및 항염활성이 뛰어난 노루참나물 추출물을 제공하는데 목적이 있다.An object of the present invention is to provide a roe deer extract having excellent whitening activity and excellent antioxidant and anti-inflammatory activity.

또한, 본 발명의 노루참나물 추출물을 유효성분으로 포함하는 조성물을 제공하는데 목적이 있다.In addition, an object of the present invention is to provide a composition comprising the roe deer extract of the present invention as an active ingredient.

또한, 본 발명의 노루참나물 추출물로부터 분리한 카페익산 메틸 에스테르를 제공하고, 이 분리된 물질을 유효성분으로 포함하는 조성물을 제공하는데 그 목적이 있다.It is also an object of the present invention to provide a caffeic acid methyl ester separated from the roe deer extract of the present invention, and to provide a composition comprising the separated substance as an active ingredient.

본 발명은 미백활성을 나타내는 노루참나물 추출물에 관한 것이다.The present invention relates to a roe deer extract extract showing whitening activity.

본 발명의 노루참나물 추출물은 노루참나물을 세척한 후 3일 동안 40 ℃로 열풍건조한 다음 분쇄하고, 이 노루참나물 분쇄물을 70 % 에탄올에 침적시키고 2 ~ 3 일동안 실온에서 교반하여 침출한 다음, 여과기로 여과하여 조추출물을 제조하고, 이 조추출물을 극성이 다른 용매인 n- 헥산, 에틸아세테이트, 부탄올을 이용해 순차적으로 분획하여 추출분획물을 제조하는 것으로 구성된다.The roe deer extract of the present invention is dried after rinsing the roe roe sprout at 40 ° C. for 3 days, and then pulverized, and immersing the roe deer pulverized product in 70% ethanol and stirring at room temperature for 2-3 days, The crude extract was prepared by filtration with a filter, and the crude extract was fractionated sequentially using solvents having different polarities, n-hexane, ethyl acetate and butanol, to prepare an extract fraction.

또한, 본 발명은 상기의 노루참나물 추출물이 유효성분으로 포함된 조성물을 제조하는 것으로 구성된다.In addition, the present invention consists in preparing a composition comprising the roe deer extract as an active ingredient.

또한, 본 발명은 노루참나물 추출물로부터 분리한 카페익산 메틸 에스테르가 유효성분으로 포함된 조성물을 제조하는 것으로 구성된다.In addition, the present invention consists in preparing a composition comprising the caffeic acid methyl ester isolated from the roe deer extract as an active ingredient.

한편, 본 발명의 노루참나물(Pimpinella komarovii)은 산형과에 속하는 다년생초본으로서 제주도를 비롯하여 우리나라 북부, 함경남도, 동부시베리아 등지에 자생하고 있으며, 꽃은 8월에 백색으로 피고, 잎은 호생하고 2회 3출 복엽이며, 근엽은 성장 후 고사하며, 열매는 좁은 난형으로 밋밋하고 털이 없는 식물이다.On the other hand, the roe deer ( Pimpinella komarovii ) of the present invention is a perennial herb belonging to the mountain family, native to Jeju Island, North Korea, Hamgyongnam-do, East Siberia, etc., and the flowers bloom white in August, and the leaves are revived twice. It is a three-lobed lobe, the rosettes die after growth, and the fruit is a narrow, ovoid, flat, hairless plant.

현재 노루참나물(Pimpinella komarovii)에 대한 연구보고는 없고, 다만 참나물속 (Pimpinella) 중 몇몇 종들에서 기관지 천식 및 위장장애 치료제, 구풍제, 진경제, 거담제, 진위제 등의 약재 및 향신료와 식용 등으로 쓰인다고 알려져 있다.There are currently no studies on Pimpinella komarovii , but some of the species of Pimpinella are known to be used for the treatment of bronchial asthma and gastrointestinal disorders, as a repellent, antifungal, antispasmodic, expectorant, etc. have.

본 발명의 발명자들이 미백 활성 등 생리활성이 뛰어난 천연식물 및 천연식물에서 분리된 물질에 대한 연구를 하던 중 노루참나물의 추출물 및 그로부터 분리한 단일물질에서 멜라닌 합성억제 활성 및 티로시나제 저해활성 등의 미백활성이 뛰어나고 항산화 및 항염활성까지 그 생리활성이 뛰어나다는 사실을 알게 되어 본 발명을 완성하였다.While the inventors of the present invention have studied natural plants having excellent physiological activity such as whitening activity and substances isolated from natural plants, whitening activity such as melanin synthesis inhibitory activity and tyrosinase inhibitory activity in extracts of roe deer sprout and single substance separated therefrom The present invention was completed by knowing that the physiological activity is excellent and excellent in antioxidant and anti-inflammatory activity.

본 발명의 노루참나물(Pimpinella komarovii) 추출물이 melan-a 세포주의 세포 생존률에 미치는 영향을 알아보기 위하여 세포독성 실험을 한 결과, 대조군의 세포 생존률을 100 %로 하였을 때, 노루참나물의 조추출물 농도 중 12.5, 25 ㎍/㎖ 농도에서는 각각 96 %, 97 %로 대조군에 비해 약간 감소하였으나, 50 ㎍/㎖ 농도에서 102 %, 100 ㎍/㎖ 농도에서 107 %로 대조군과 비슷하였다(도 11).Roe deer yam of the present invention ( Pimpinella) To determine the effect of komarovii ) extract on the cell viability of melan-a cell line, 12.5, 25 ㎍ / mL concentration of crude extract of roe deer sprout when the cell viability of control group was 100%. At 96% and 97% respectively, slightly decreased compared to the control, but 102% at 50 ㎍ / mL concentration and 107% at 100 ㎍ / mL concentration, similar to the control (Fig. 11).

이처럼 대조군에 비해 약간의 차이가 있으나 유의할만한 변화를 나타내지 않았다.As such, there was a slight difference compared to the control group, but there was no significant change.

또한, 본 발명의 노루참나물 추출물에 대하여 미백활성을 측정하기 위해 멜라닌 생성량을 측정하고, 티로시나제 활성에 대한 억제율을 측정하였다.In addition, the melanin production amount was measured to measure the whitening activity of the roe deer extract of the present invention, and the inhibition rate against tyrosinase activity was measured.

본 발명의 노루참나물 조추출물을 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖까지 다양한 농도로 3 일 동안 처리한 후, 멜라닌 생성량을 측정한 결과, 대조군인 melazove 30 uM((주) 태평양) 처리시 77 %의 멜라닌 생성 억제활성을 보였으며, 각각 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖ 농도까지 처리시 11 %, 23 %, 42 %, 62 %로 억제활성을 보였다(도12A). The crude extract of the present invention was treated with various concentrations up to 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml for 3 days, and then the melanin production was measured. As a control, melazove 30 uM (Pacific Co., Ltd.) showed 77% melanogenesis inhibitory activity, and 11%, 23%, 42 when treated to concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml, respectively. %, 62% showed inhibitory activity (FIG. 12A).

따라서, 노루참나물 조추출물은 melan-a 세포에서 투여량이 처리 농도가 증가 함에 따라 멜라닌 생성이 억제되는 경향을 보였으며, 통계학적으로 유의성이 있음을 보여주었으며, 노루참나물 추출분획물에서는 에틸아세테이트 분획물이 48 %로 가장 높은 효과를 보였다(도 12B).Therefore, the crude extract of roe deer bran showed a tendency to inhibit melanin production as the dose was increased in melan-a cells, and it was statistically significant. The ethyl acetate fraction was 48% in the roe deer extract extract. The highest effect was shown in% (FIG. 12B).

또한, 노루참나물 추출물의 처리 후 최종 멜라닌양이 억제된 것은 멜라닌 생 성 및 합성에 관여하는 효소들의 활성과 관련이 있음을 나타내므로 melan-a 세포에서 티로시나제(tyrosinase) 활성을 측정하였다.In addition, the inhibition of the final melanin amount after the treatment of the roe deer extract was measured tyrosinase activity in melan-a cells because it indicates that it is related to the activity of enzymes involved in melanogenesis and synthesis.

그 결과, melazove 30 uM((주) 태평양) 처리시 75 %의 티로시나제 억제활성을 보였으며, 노루참나물 조추출물을 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖ 농도 처리시 각각 45 %, 59 %, 66 %, 74 %의 억제활성을 보였다(도 13A).As a result, it showed 75% tyrosinase inhibitory activity when treated with melazove 30 uM (Pacific Co., Ltd.), and 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL concentrations of roe deer extract 45%, 59%, 66%, 74% of inhibitory activity, respectively (FIG. 13A).

따라서, 노루참나물 조추출물은 melan-a 세포에서 처리농도가 증가함에 따라 티로시나제 활성도가 억제되는 경향을 보았으며, 통계학적으로 유의성이 있음을 보여 주었고, 노루참나물의 추출분획물은 에틸아세테이트 분획물에서 25 ㎍(74 %), 50 ㎍(78 %)의 높은 억제효과를 보였다(도 13B). Therefore, the crude extracts of roe deer sprouts showed a tendency of inhibiting tyrosinase activity with increasing treatment concentration in melan-a cells and showed statistical significance. The extract fraction of roe deer sprouts was 25 ㎍ in ethyl acetate fraction. (74%), 50 ㎍ (78%) showed a high inhibitory effect (Fig. 13B).

또한, 본 발명의 노루참나물 추출물에 대한 항산화활성을 실험하였다.In addition, the antioxidant activity of the roe deer extract of the present invention was tested.

항산화 물질의 가장 특이적인 기작은 유리기와 반응하는 것으로 유리기 소거작용은 활성라디칼(free radical)에 전자를 공여하여 식물 중의 항산화 효과나 인체에서 노화를 억제하는 척도로 사용되므로 전자공여능(electron donating ability)측정에 의한 DPPH 자유라디칼 소거활성을 측정하였다.The most specific mechanism of antioxidants reacts with free radicals. The free radical scavenging action is used as a measure of antioxidant activity in plants or as a measure of inhibiting aging in the human body by donating electrons to free radicals. DPPH free radical scavenging activity was measured by measurement.

그 결과, 상용화된 아스코르브산(ascorbic acid)의 IC50은 8.23 ㎍으로 높은 활성라디칼 억제효과를 보였다. As a result, IC 50 of commercialized ascorbic acid showed 8.23 ㎍ with high active radical inhibition effect.

노루참나물 조추출물의 IC50은 227.32 ㎍으로 낮은 효과를 보였으나, 노루참나물 에틸아세테이트 분획물은 IC50이 79.76 ㎍, 부탄올 분획물은 IC50이 161.33 ㎍/㎖)층은 조추출물보다 2 배 이상 활성산소 억제 효과를 보였다. And showed an IC 50 is low effect 227.32 ㎍ of roe chamnamul crude extract, deer chamnamul ethyl acetate fraction IC 50 is 79.76 ㎍, butanol fraction is the IC 50 161.33 ㎍ / ㎖) layer is twice or more active oxygen than the crude extract It showed an inhibitory effect.

또, 노루참나물 추출물의 nitric oxide 생성저해 활성을 측정한 결과, 노루참나물 조추출물과 물분획, 에틸아세테이트 분획물, 부탄올 분획물에서는 저해율이 매우 낮았으나, 헥산 분획물에서는 IC50이 123.53으로 억제효과를 보였다.Inhibition of nitric oxide production of roe deer extract showed that the crude extract, water fraction, ethyl acetate fraction and butanol fraction showed very low inhibition rate, but IC 50 was 123.53 in hexane fraction.

또, 잔틴 산화효소 억제 및 과산화물 소거활성을 측정한 결과, 노루참나물 에탄올 조추출물을 포함한 분획에서의 잔틴 산화효소(xanthine oxidase) 억제효과는 매우 낮았으나, 과산화물 소거활성은 노루참나물 조추출물은 IC50가 30.79 ㎍/㎖, 에틸아세테이트 분획물은 IC50이 8.97 ㎍/㎖, 부탄올 분획물의 IC50은 14.03 ㎍/㎖, 헥산분획물의 IC50은 151.33 ㎍/㎖, 물 분획물은 IC50이 >400 ㎍/㎖으로서 에틸아세테이트 분획물에서 높은 억제효과를 나타내었다(표 4). In addition, Xanthine oxidase inhibition and peroxide scavenging activity were measured to show that the xanthine oxidase inhibitory effect was very low in the fraction containing ethanol crude ethanol crude extract, but the peroxide scavenging activity was 50 30.79 µg / ml, the ethyl acetate fraction is IC 50 at 8.97 µg / ml, butanol fraction IC 50 at 14.03 µg / ml, hexane fraction IC 50 at 151.33 µg / ml, and water fraction at IC 50 > 400 µg / ml. It showed a high inhibitory effect on the ethyl acetate fraction as ml (Table 4).

따라서, 본 발명의 노루참나물 추출물이 항산화 활성이 뛰어나다는 사실을 알 수 있었다.Therefore, it was found that the roe deer extract of the present invention is excellent in antioxidant activity.

또한, 본 발명의 노루참나물 추출물에 대하여 RAW264.7cell에서의 Cell NO 억제효과 및 세포독성저해 효과를 측정하여 항염활성을 실험한 결과, IC50값은 75 ㎍으로서 비교적 높은 억제효과를 보였다(도 14A).In addition, the anti-inflammatory activity was measured by measuring the cell NO inhibitory effect and cytotoxicity inhibitory effect in RAW264.7cell with respect to the roe deer extract of the present invention, IC 50 value was 75 ㎍ showed a relatively high inhibitory effect (Fig. 14A). ).

또한, MTT assay 결과 100 ㎍/㎖처리까지 세포독성은 없었다(도 14B). In addition, MTT assay showed no cytotoxicity until 100 ㎍ / ㎖ treatment (Fig. 14B).

한편, 본 발명의 노루참나물 추출물로부터 분리된 단일 물질은 LC-MS를 통하여 분자량이 194 인 물질임을 확인하였다(도 6).On the other hand, the single material separated from the roe deer extract of the present invention was confirmed that the molecular weight of 194 through LC-MS (Fig. 6).

또한, 1H-NMR에서 δ 7.54 (d, J=15.8Hz, H-8), 7.02 (d, J=1.9Hz, H-2), 6.93 (dd, J=8.3, 1.9Hz, H-5), 6.76 (d, 8.3Hz, H-6), 6.25 (d, 15.8, H-7), 3.75 (s, OMe)이고(도 7), 13C-NMR은 δ 169.93 (C-9), 149.99 (C-4), 147.12 (C-7), 147.06 (C-4), 127.66 (C-1), 123.03 (C-6), 116.59 (C-5), 115.12 (C-2), 114.79 (C-8), 51.97 (OMe)이며(도 8), H-H COSY에서 8번 H와 7번 H가 Coupling되어 있고, 5번 H와 6번 H가 Coupling 되어 있다(도 9). Further, in 1 H-NMR, δ 7.54 (d, J = 15.8 Hz, H-8), 7.02 (d, J = 1.9 Hz, H-2), 6.93 (dd, J = 8.3, 1.9 Hz, H-5 ), 6.76 (d, 8.3 Hz, H-6), 6.25 (d, 15.8, H-7), 3.75 (s, OMe) (FIG. 7), and 13 C-NMR is δ 169.93 (C-9), 149.99 (C-4), 147.12 (C-7), 147.06 (C-4), 127.66 (C-1), 123.03 (C-6), 116.59 (C-5), 115.12 (C-2), 114.79 (C-8) and 51.97 (OMe) (FIG. 8), H and CO are Huped 8 and 7, and H and 6H are coupled in HH COSY (FIG. 9).

상기 분석결과 본 발명의 노루참나물 추출물로부터 분리한 물질은 카페익산 메틸 에스테르(Caffeic acid methyl ester)로 동정되었다(도 10). As a result of the analysis, the material isolated from the roe deer extract of the present invention was identified as Caffeic acid methyl ester (FIG. 10).

따라서, 본 발명의 노루참나물 추출물로부터 분리된 단일 물질이 카페익산 메틸 에스테르로 밝혀졌기 때문에 이후 노루참나물 유래 카페익산 메틸 에스테르를 대량으로 분리하는 경우에는 통상적인 다양한 방법을 이용하여 분리가능하다.Therefore, since a single substance isolated from the roe deer sprout extract of the present invention was found to be caffeic acid methyl ester, it can be separated using a variety of conventional methods when separating the roe deer sprout-derived caffeic acid methyl ester in large quantities.

한편, 본 발명의 노루참나물 추출물은 식품첨가제, 사료첨가제, 음료조성물, 화장료조성물 등으로 다양하게 활용할 수 있다.On the other hand, the roe deer extract of the present invention can be utilized in various ways, such as food additives, feed additives, beverage compositions, cosmetic compositions.

본 발명의 노루참나물 추출물 및 이로부터 분리된 카페익산 메틸 에스테르를 활성성분으로 포함하는 미백용 조성물을 제조할 수 있다.The whitening composition comprising the roe deer extract of the present invention and the caffeic acid methyl ester separated therefrom as an active ingredient can be prepared.

또한, 본 발명은 상기와 같은 생리활성을 나타내는 노루참나물 추출물 및 이로부터 분리된 카페익산 메틸 에스테르를 유효성분으로 포함하는 염증 예방 및 개선용 조성물 또는 항산화용 조성물을 제조할 수 있다.In addition, the present invention can prepare a composition for preventing or improving inflammation or an antioxidant comprising a roe deer sprout extract showing the physiological activity as described above and a caffeic acid methyl ester separated therefrom as an active ingredient.

이때, 본 발명의 조성물은 환, 캡슐, 과립, 현탁액 등 다양한 형태로 제조될 수 있다.At this time, the composition of the present invention may be prepared in various forms such as pills, capsules, granules, suspensions.

한편, 본 발명의 노루참나물 추출물 및 이로부터 분리된 카페익산 메틸 에스테르가 유효성분으로 포함된 조성물 제조시, 첨가량은 조성물의 용도 및 형태에 다양하게 조절할 수 있다.On the other hand, when preparing a composition containing the roe deer extract of the present invention and the caffeic acid methyl ester separated therefrom as an active ingredient, the addition amount can be variously adjusted according to the use and form of the composition.

이하, 본 발명의 노루참나물 추출물 및 이로부터 분리한 물질에 대하여 실시예 및 실험예를 통하여 상세히 설명하나, 이들이 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the roe deer extract of the present invention and the material separated therefrom will be described in detail through Examples and Experimental Examples, but these are not intended to limit the scope of the present invention.

<실시예 1> 본 발명의 노루참나물 추출물 제조<Example 1> roe deer extract extract of the present invention

제주도에서 자생하고 있는 노루참나물(Pimpinella komarovii)을 채집하여 준비하였다. Pimpinella komarovii , native to Jeju Island, was collected and prepared.

준비한 노루참나물 전초(全草)를 흐르는 물에 세척한 후 3 일 동안 40 ℃로 열풍건조하였다.The prepared roe deer starch outpost (全 草) was washed with running water and hot-air dried at 40 ° C. for 3 days.

건조된 노루참나물 전초 100 g을 분쇄기로 분쇄하고, 이 분쇄물을 70 % 에탄올에 침적시키고 2 ~ 3 일 동안 실온에서 교반하여 침출하였다. 100 g of dried roe deer outpost were crushed with a grinder, which was immersed in 70% ethanol and stirred at room temperature for 2-3 days.

이 침출물을 여과기로 여과하고, 상기의 침출 및 여과과정을 2 ~ 3 회 더 반복하여 본 발명의 노루참나물 조추출물을 제조하였다.The leachate was filtered with a filter, and the leaching and filtration of the present invention was prepared by repeating the above leaching and filtration two to three times.

상기의 노루참나물 조추출물 10 g을 증류수에 용해시키고, n-헥산을 첨가하여 n-헥산층과 수층으로 분획한 후 이를 2 회 반복하여 헥산층을 얻었다. 10 g of the crude roe deer extract was dissolved in distilled water, n-hexane was added thereto, fractionated into an n-hexane layer and an aqueous layer, and this was repeated twice to obtain a hexane layer.

그 다음, 상기의 수층에 에틸아세테이트를 첨가하여 에틸아세테이트와 수층 으로 분획한 후 이를 3 회 반복하여 에틸아세테이트 층을 얻었고, 상기 수층에 부탄올을 첨가하여 부탄올 층과 수층을 분획한 후 이를 3 회 반복하여 부탄올층을 얻었다. Then, ethyl acetate was added to the aqueous layer, the mixture was partitioned into ethyl acetate and the aqueous layer, and then repeated three times to obtain an ethyl acetate layer. Butanol was added to the aqueous layer to separate the butanol layer and the aqueous layer and then repeated three times. To obtain a butanol layer.

상기의 분획물을 각각 감압농축시켜 노루참나물 헥산분획물 1.1 g, 에틸아세테이트 분획물 3.0 g, 부탄올 분획물 3.4 g, 물분획물 2.5 g을 제조하였다.The fractions were concentrated under reduced pressure, respectively, to prepare 1.1 g of roe deer hexane fraction, 3.0 g of ethyl acetate fraction, 3.4 g of butanol fraction, and 2.5 g of water fraction.

<실험예 1> 본 발명의 노루참나물 추출물로부터 단일 물질의 분리실험<Experimental Example 1> Separation experiment of a single substance from the roe deer extract of the present invention

본 발명의 실시예 1에서 제조된 노루참나물 조추출물 및 추출분획물을 준비하였다.Crude extract and extract fractions of the roe deer sprout prepared in Example 1 of the present invention were prepared.

준비한 조추출물 및 추출분획물에 대하여 고속액체크로마토그래피(HPLC) 분석을 하였다.High performance liquid chromatography (HPLC) analysis was performed on the crude extracts and extracts.

그 분석조건은 다음과 같이 하였다.The analysis conditions were as follows.

<표 1> 노루참나물 분석시 HPLC 장치 조건 <Table 1> HPLC apparatus conditions for the analysis of roe deer sprouts

HPLCHPLC Waters Alliance 2695 systemWaters Alliance 2695 system 컬럼(Column)Column SunfireTM C18 5μm 4.6×250mmSunfire TM C18 5μm 4.6 × 250mm 검출기(Detecter)Detecter 광다이오드 어레이(Photodiode Array) 검출기Photodiode Array Detector 유량(Flow rate)Flow rate 1.0 ㎖/min1.0 ml / min 주입량(Injection Volume)Injection Volume 10 ㎕10 μl 컬럼온도(Column Temperature)Column temperature 25 ℃25 샘플온도(Sample Temperature)Sample Temperature 10 ℃10 ℃

<표 2> HPLC 기울기용리를 위한 이동상 조건TABLE 2 Mobile phase conditions for HPLC gradient elution

Program orderProgram order TimeTime FlowFlow H2OH2O AcetonitrileAcetonitrile CurveCurve 1One 1.001.00 95.095.0 5.05.0 22 5.005.00 1.001.00 95.095.0 5.05.0 66 33 10.0010.00 1.001.00 90.090.0 10.010.0 66 44 30.0030.00 1.001.00 80.080.0 20.020.0 66 55 40.0040.00 1.001.00 80.080.0 20.020.0 66 66 50.0050.00 1.001.00 70.070.0 30.030.0 66 77 60.0060.00 1.001.00 70.070.0 30.030.0 66 88 70.0070.00 1.001.00 50.050.0 50.050.0 66 99 80.0080.00 1.001.00 50.050.0 50.050.0 66 1010 81.0081.00 1.001.00 0.00.0 100.0100.0 66 1111 90.0090.00 1.001.00 0.00.0 100.0100.0 66 1212 91.0091.00 1.001.00 95.095.0 5.05.0 66 1313 100.00100.00 1.001.00 95.095.0 5.05.0 66

그 결과, 도 2에서 보는 바와 같이, 46 분대에 나타내는 피크 1을 분리하여 기준물질로 표기하였다.As a result, as shown in FIG. 2, the peak 1 shown in 46 components was isolate | separated and described as a reference substance.

본 발명의 노루참나물 추출물과 각 추출분획물에 대하여 HPLC 크로마코그래피를 통하여 에틸아세테이트 분획물층에서 상기의 물질이 응집되었음을 확인하였다(도 3).It was confirmed that the material was aggregated in the ethyl acetate fraction layer through HPLC chromatography for the roe deer extract and each extract fraction of the present invention (FIG. 3).

그러나, 부탄올 분획물층에서는 피크 1이 RT(머무름시간) 46 분에서 아주 미약하게 나타났다(도 4).However, in the butanol fraction layer, peak 1 was very weak at 46 min RT (FIG. 4).

따라서, 에틸아세테이트층을 Prep-LC를 이용하여 전개용매 H2O/Acetonitril (90/10)을 기울기로 50 분 동안 용리하여 순수 피크 1을 얻었다.Thus, the ethyl acetate layer was eluted with Prep-LC for 50 minutes at a solvent H 2 O / Acetonitril (90/10) with a gradient to obtain pure peak 1.

또한, 단일 물질을 HPLC로 분석하여 순도를 확인하였다(도 5).In addition, single material was analyzed by HPLC to confirm purity (FIG. 5).

<실험예 2> 본 발명의 노루참나물 추출물로부터 분리한 단일 물질에 대한 분석실험Experimental Example 2 Analysis of a Single Substance Isolated from the Roe Deer Sprout Extract of the Present Invention

실험예 1에서 분리한 단일 물질에 대하여 분자량 확인을 위해 LC-MS(ESI-MS, m/z 193[M-H])을 이용하여 negative로 확인한 결과, 분리한 물질의 분자량이 194 임을 확인하였다(도 6). As a result of negative confirmation using LC-MS (ESI-MS, m / z 193 [MH]) to confirm the molecular weight of the single substance separated in Experimental Example 1, it was confirmed that the molecular weight of the separated substance was 194 (Fig. 6).

또한, NMR 분석에서 1H-NMR(CD3OD, 400MHz)을 확인 한 결과 7.54ppm(d, J=15.8Hz, H-8), 6.25ppm(d, 15.8, H-7)에서 trans구조를 갖고 있음을 확인할 수 있고, 7.02ppm(d, J=1.9Hz, H-2), 6.93ppm(dd, J=8.3, 1.9Hz, H-5), 6.76ppm(d, 8.3Hz, H-6)에서는 benzene ring, 3.75ppm(s, OMe)에서 singlet은 methyl기에 전기음성도가 큰 O원자가 붙어 있는 형태라고 예상할 수 있었다(도 7). In addition, 1 H-NMR (CD 3 OD, 400 MHz) was confirmed by NMR analysis. As a result, trans structures were obtained at 7.54 ppm (d, J = 15.8 Hz, H-8) and 6.25 ppm (d, 15.8, H-7). It can be confirmed that it has, 7.02ppm (d, J = 1.9Hz, H-2), 6.93ppm (dd, J = 8.3, 1.9Hz, H-5), 6.76ppm (d, 8.3Hz, H-6 In the benzene ring, 3.75ppm (s, OMe), singlet can be expected to have a form of O atom with a large electronegativity attached to a methyl group (FIG. 7).

13C-NMR(CD3OD, 100MHz)을 확인 한 결과 169.93ppm(C-9)에서 ester기가 있음을 확인하였고, 149.99ppm(C-4), 147.06ppm(C-3), 127.66ppm(C-1), 123.03ppm(C-6), 116.59ppm(C-5), 115.12ppm(C-2)에서 방향족 형태라 예측되었다(도 8). 13 C-NMR (CD 3 OD, 100MHz) confirmed that the ester group at 169.93ppm (C-9), 149.99ppm (C-4), 147.06ppm (C-3), 127.66ppm (C -1), 123.03ppm (C-6), 116.59ppm (C-5), 115.12ppm (C-2) were predicted as aromatic form (Fig. 8).

또한, 51.97ppm(OMe)에서 O원자가 붙은 methyl기의 C임을 확인할 수 있었다(도 8).In addition, it was confirmed that the C group of the methyl group attached to the O atom at 51.97 ppm (OMe) (Fig. 8).

따라서, 이를 정리하면 1H-NMR에서 δ 7.54 (d, J=15.8Hz, H-8), 7.02 (d, J=1.9Hz, H-2), 6.93 (dd, J=8.3, 1.9Hz, H-5), 6.76 (d, 8.3Hz, H-6), 6.25 (d, 15.8, H-7), 3.75 (s, OMe)이고, 13C-NMR은 δ 169.93 (C-9), 149.99 (C-4), 147.12 (C-7), 147.06 (C-4), 127.66 (C-1), 123.03 (C-6), 116.59 (C-5), 115.12 (C-2), 114.79 (C-8), 51.97 (OMe)이다. Thus, in summary, at 1 H-NMR, δ 7.54 (d, J = 15.8 Hz, H-8), 7.02 (d, J = 1.9 Hz, H-2), 6.93 (dd, J = 8.3, 1.9 Hz, H-5), 6.76 (d, 8.3 Hz, H-6), 6.25 (d, 15.8, H-7), 3.75 (s, OMe), and 13 C-NMR is δ 169.93 (C-9), 149.99 (C-4), 147.12 (C-7), 147.06 (C-4), 127.66 (C-1), 123.03 (C-6), 116.59 (C-5), 115.12 (C-2), 114.79 ( C-8), 51.97 (OMe).

H-H COSY에서 8번 H와 7번 H가 Coupling되어 있고, 5번 H와 6번 H가 Coupling 되어 있음을 확인할 수 있었다(도 9). In H-H COSY it was confirmed that 8 H and 7 H are coupling, and 5 H and 6 H are coupling (FIG. 9).

따라서, 위 결과를 정리하면 아래의 표 3과 같이 본 발명의 노루참나물 추출물로부터 분리한 물질은 카페익산 메틸 에스테르(Caffeic acid methyl ester)로 동정하였다(도 10). Therefore, summarizing the above results, as shown in Table 3 below, the material separated from the roe deer extract of the present invention was identified as caffeic acid methyl ester (Caffeic acid methyl ester) (FIG. 10).

한편, 위 물질은 담쟁이덩굴(Parthenocissue tricuspidata)에서 보고한 결과(Saleem et al, 2004)와 동일하여 카페익산 메틸 에스테르(Caffeic acid methyl ester)임을 확인하였다.On the other hand, the above substance was confirmed in the same as the results reported by the ivy (Parthenocissue tricuspidata) (Saleem et al, 2004) Caffeic acid methyl ester (Caffeic acid methyl ester).

<표 3> NMR 분석결과 데이터<Table 3> NMR Analysis Result Data

  CC HH   1One 127.66127.66     22 115.12115.12 7.027.02 (1H, d, 1.9Hz)(1H, d , 1.9 Hz) 33 147.06147.06     44 149.99149.99     55 116.59116.59 6.936.93 (1H, dd, 8.3,1.9Hz)(1H, dd , 8.3,1.9 Hz) 66 123.03123.03 6.766.76 (1H, d, 8.3Hz)(1H, d , 8.3 Hz) 77 147.12147.12 6.256.25 (1H, d, 15.8Hz)(1H, d , 15.8 Hz) 88 114.79114.79 7.547.54 (1H, d, 15.8Hz)(1H, d , 15.8 Hz) 99 169.93169.93     O-MeO-Me 51.9751.97 3.753.75 (3H, s)(3H, s )

<실시예 2> 노루참나물 유래 카페익산 메틸 에스테르 물질의 대량생산Example 2 Mass Production of Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

본 발명의 실시예 1의 방법으로 제조된 노루참나물 조추출물을 준비하였다.The roe deer crude crude extract prepared by the method of Example 1 of the present invention was prepared.

노루참나물 유래 카페익산 메틸 에스테르 물질을 대량으로 분리하기 위하여 먼저 glass column에 sephadex LH-20을 충진시키고 준비한 조추출물 1 ㎏을 넣었다.In order to separate a large amount of caffeic acid methyl ester derived from roe deer sprouts, sephadex LH-20 was first charged into a glass column and 1 kg of the crude extract was added.

여기에 메탄올/디클로로메탄(30/70)을 흘려주어 카페익산 메틸 에스테르의 분자량(194) 크기로 분리하였다.Methanol / dichloromethane (30/70) was flowed here and it isolate | separated into the molecular weight (194) size of the caffeic acid methyl ester.

그 후 중간압력 액체 크로마토그래피(MPLC)를 이용하여 전개용매 메탄올과 디클로로메탄을 1 : 3 비율로 100 분 동안 흘려주어 카페익산 메틸 에스테르 물질 5 g을 분리하였다.Thereafter, 5 g of the caffeic acid methyl ester material was separated by flowing the developing solvent methanol and dichloromethane at a ratio of 1: 3 for 100 minutes by using medium pressure liquid chromatography (MPLC).

<실시예 3> 노루참나물 추출물을 이용한 환 제조 <Example 3> Manufacture of pills using roe deer extract

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 동결건조 한 후 분말로 준비하였다.After the lyophilized roe deer extract extract prepared by the method of Example 1 of the present invention was prepared as a powder.

준비한 분말 1 ㎏에 물 200 g, 꿀 100 g을 넣고 반죽하여 환 제조장치에 넣어 환 모양으로 만든 후, 건조기에 넣고 30 ℃ 에서 12 시간 동안 건조하여 환을 제조하였다.200 g of water and 100 g of honey were put in 1 kg of the prepared powder, kneaded, put into a pill manufacturing apparatus to make a ring shape, and then put into a drier and dried at 30 ° C. for 12 hours to prepare a ring.

<실시예 4> 노루참나물 유래 카페익산 메틸 에스테르를 이용한 캡슐 제조Example 4 Preparation of Capsules Using Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

본 발명의 실시예 2의 방법에 의해 분리된 카페익산 메틸 에스테르를 준비하였다.The caffeic acid methyl ester isolated by the method of Example 2 of the present invention was prepared.

준비한 물질을 이용해 통상적인 방법으로 캡슐로 제조하였다.Capsules were prepared in a conventional manner using the prepared material.

<실시예 5> 노루참나물 추출물이 함유된 음료조성물의 제조Example 5 Preparation of a Beverage Composition Containing Extract of Roe Deer Sprouts

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 준비하였다.A roe deer extract was prepared by the method of Example 1 of the present invention.

정제수 1 ℓ 당 준비한 노루참나물 추출물 20 g, 비타민 B2 4 g, 비타민 B6 6 g, 니코틴산아미드 10 g, 구연산 30 g, 액상과당 2 ㎏, 허브향 70 ㎖ 를 첨가하여 본 발명의 노루참나물 추출물이 함유된 음료조성물을 제조하였다.20 g of roe deer extract, 1 g of vitamin B2, 6 g of nicotinic acid amide, 10 g of citric acid amide, 30 g of citric acid, 2 kg of liquid fructose, and 70 ml of herbal flavor were added to the extract of the roe deer extract of the present invention. Prepared beverage composition.

<실시예 6> 노루참나물 유래 카페익산 메틸 에스테르가 함유된 음료조성물의 제조Example 6 Preparation of a Beverage Composition Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

본 발명의 실시예 2의 방법에 의해 분리된 노루참나물 유래 카페익산 메킬 에스테르를 준비하였다.The roe deer sprout-derived caffeic acid methyl ester isolated by the method of Example 2 of the present invention was prepared.

정제수 1 ℓ 당 준비한 물질 10 g, 비타민 B2 4 g, 비타민 B6 6 g, 니코틴산아미드 10 g, 구연산 30 g, 액상과당 2 ㎏, 허브향 70 ㎖ 를 첨가하여 본 발명의 노루참나물 유래 카페익산 메틸 에스테르가 함유된 음료조성물을 제조하였다.10 g of prepared substance per 1 liter of purified water, 4 g of vitamin B2, 6 g of vitamin B6, 10 g of nicotinic acid amide, 30 g of citric acid, 2 kg of liquid fructose, and 70 ml of herb flavor were added to the caffeic acid methyl ester derived from roe deer sprout of the present invention. A beverage composition containing was prepared.

<실시예 7> 노루참나물 추출물이 함유된 미백용 화장료조성물의 제조<Example 7> Preparation of the whitening cosmetic composition containing the roe deer extract

1. 미백용 스킨조성물 제조1. Preparation of skin composition for whitening

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 준비하였다.A roe deer extract was prepared by the method of Example 1 of the present invention.

통상적인 스킨조성물 제조시, 상기의 추출물을 2 중량% 첨가하여 미백용 스킨조성물을 제조하였다.In preparing a conventional skin composition, 2% by weight of the extract was added to prepare a skin composition for whitening.

2. 미백용 로션조성물 제조2. Manufacture lotion composition for whitening

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 준비하였다.A roe deer extract was prepared by the method of Example 1 of the present invention.

통상적인 로션조성물 제조시, 상기의 추출물을 2 중량% 첨가하여 미백용 로 션조성물을 제조하였다.In preparing a conventional lotion composition, 2 wt% of the extract was added to prepare a lotion composition for whitening.

<실시예 8> 노루참나물 유래 카페익산 메틸 에스테르가 함유된 미백용 화장료조성물의 제조Example 8 Preparation of a Whitening Cosmetic Composition Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

1. 미백용 크림 제조1. Cream for whitening

본 발명의 실시예 2의 노루참나물 유래 카페익산 메틸 에스테르를 준비하였다.A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

통상적인 크림 조성물 제조시, 상기의 물질 2 중량 % 첨가하여 미백용 크림조성물을 제조하였다.In preparing a conventional cream composition, 2 wt% of the above material was added to prepare a cream composition for whitening.

2. 미백용 에센스 조성물 제조2. Preparation of whitening essence composition

본 발명의 실시예 2의 노루참나물 유래 카페익산 메틸 에스테르를 준비하였다.A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

통상적인 에센스 조성물 제조시, 상기의 물질 1 중량 % 첨가하여 미백용 에센스조성물을 제조하였다.In preparing a typical essence composition, 1 wt% of the above material was added to prepare a whitening essence composition.

<실시예 9> 노루참나물 추출물이 함유된 염증성 질환의 개선 및 치료용 화장료조성물의 제조Example 9 Preparation of Cosmetic Composition for Amelioration and Treatment of Inflammatory Disease Containing Roe Deer Fructus Extract

1. 염증성 질환의 개선 및 치료용 에센스 조성물 제조1. Preparation of essence composition for improvement and treatment of inflammatory disease

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 준비하였다.A roe deer extract was prepared by the method of Example 1 of the present invention.

통상적인 에센스 조성물 제조시, 상기의 추출물을 2 중량% 첨가하여 염증성 질환의 개선 및 치료용 에센스 조성물을 제조하였다.In preparing a typical essence composition, 2% by weight of the extract was added to prepare an essence composition for improving and treating inflammatory diseases.

2. 염증성 질환의 개선 및 치료용 로션조성물 제조2. Preparation of lotion composition for improving and treating inflammatory diseases

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 준비하였다.A roe deer extract was prepared by the method of Example 1 of the present invention.

통상적인 로션조성물 제조시, 상기의 추출물을 3 중량% 첨가하여 염증성 질환의 개선 및 치료용 로션조성물을 제조하였다.In preparing a conventional lotion composition, the extract was added 3% by weight to prepare a lotion composition for improving and treating inflammatory diseases.

<실시예 10> 노루참나물 유래 카페익산 메틸 에스테르가 함유된 염증성 질환의 개선 및 치료용 화장료조성물의 제조Example 10 Preparation of Cosmetic Composition for the Improvement and Treatment of Inflammatory Disease Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

1. 염증성 질환의 개선 및 치료용 크림 제조1. Preparation of cream for improving and treating inflammatory diseases

본 발명의 실시예 2의 노루참나물 유래 카페익산 메틸 에스테르를 준비하였다.A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

통상적인 크림 조성물 제조시, 상기의 물질 3 중량% 첨가하여 염증성 질환의 개선 및 치료용 크림조성물을 제조하였다.In preparing a typical cream composition, 3 wt% of the above substance was added to prepare a cream composition for improving and treating inflammatory diseases.

2. 염증성 질환의 개선 및 치료용 에센스 조성물 제조2. Preparation of essence composition for improvement and treatment of inflammatory disease

본 발명의 실시예 2의 노루참나물 유래 카페익산 메틸 에스테르를 준비하였다.A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

통상적인 에센스 조성물 제조시, 상기의 물질 3 중량% 첨가하여 염증성 질환의 개선 및 치료용 에센스 조성물을 제조하였다.In preparing a typical essence composition, by adding 3% by weight of the above substances to prepare an essence composition for the improvement and treatment of inflammatory diseases.

<실시예 11> 노루참나물 추출물이 함유된 식품첨가제의 제조Example 11 Preparation of a Food Additive Containing Roe Deer Sprout Extract

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 동결건조 후 분말로 제조하였다.The roe deer sprout extract prepared by the method of Example 1 of the present invention was prepared as a powder after lyophilization.

통상적인 식품 제조시 상기의 분말을 2 중량% 첨가하여 사용하였다.2 wt% of the above powder was used to prepare a conventional food.

<실시예 12> 노루참나물 유래 카페익산 메틸 에스테르가 함유된 식품첨가제의 제조Example 12 Preparation of a Food Additive Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

본 발명의 실시예 2의 노루참나물 유래 카페익산 메틸 에스테르를 준비하였다.A caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

통상적인 식품 제조시 상기의 물질을 2 중량% 첨가하여 사용하였다.2 wt% of the above-mentioned substance was used to prepare a conventional food.

<실시예 13> 노루참나물 추출물이 함유된 사료첨가제의 제조Example 13 Preparation of Feed Additives Containing Roe Deer Sprout Extract

본 발명의 실시예 1의 방법에 의해 제조된 노루참나물 추출물을 동결건조 후 분말로 제조하였다.The roe deer sprout extract prepared by the method of Example 1 of the present invention was prepared as a powder after lyophilization.

통상적인 가축용 사료에 상기의 분말을 2 중량% 첨가하여 사용하였다.2 wt% of the above powder was added to a conventional livestock feed.

<실시예 14> 노루참나물 유래 카페익산 메틸 에스테르가 함유된 사료첨가제의 제조Example 14 Preparation of a Feed Additive Containing Caffeic Acid Methyl Ester Derived from Roe Deer Sprouts

본 발명의 실시예 2의 노루참나물 유래 카페익산 메틸 에스테르를 준비하였 다.Caffeic acid methyl ester derived from roe deer sprout of Example 2 of the present invention was prepared.

통상적인 가축용 사료에 상기의 물질을 2 중량% 첨가하여 사용하였다.2 wt% of the above material was added to a conventional livestock feed.

<실험예 3> 본 발명의 노루참나물 추출물에 대한 세포독성 실험<Experiment 3> Cytotoxicity test on the roe deer extract of the present invention

1. 재료준비1. Material Preparation

본 발명에 사용된 melan-a 세포는 비종양 형성 마우스의 멜라노사이트 세포주(non-tumorigenic mouse melanocyte cell line)로 C57BL의 배아(embryo)로부터 유래한 세포이다. The melan-a cells used in the present invention are cells derived from the embryo of C57BL as a non-tumorigenic mouse melanocyte cell line of non-tumorigenic mice.

melan-a 세포를 10 % 우태혈청(FBS, Gibco), 1 % 항생제-항진균제(Antibiotic-Antimycotic, Gibco), 100nM TPA, L-글루타민(L-glutamine)와 중탄산나트륨(sodium bicarbonate)이 함유된 PRMI 1640 배지(Gibco)를 사용하여 37℃, 10% CO2 세포배양기에서 배양하였다.melan-a cells were treated with PRMI containing 10% fetal bovine serum (FBS, Gibco), 1% Antibiotic-Antimycotic (Gibco), 100nM TPA, L-glutamine and sodium bicarbonate 37 ° C., 10% CO 2 using 1640 medium (Gibco) Cultured in a cell culture phase.

2. 세포독성 실험2. Cytotoxicity Experiment

본 발명의 노루참나물(Pimpinella komarovii) 추출물이 melan-a 세포주의 세포 생존률에 미치는 영향을 알아보기 위하여 세포독성 실험을 하였다.Roe deer yam of the present invention ( Pimpinella) To evaluate the effect of komarovii ) extract on cell viability of melan-a cell line, cytotoxicity experiments were performed.

melan-a 세포를 24 well plate에 well당 1×105개의 세포를 접종하고 24 시간동안 37 ℃, 10 % CO2 세포배양기에서 배양한 후 노루참나물 추출물을 각각의 well에 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖의 농도로 3 일간 처리하 여 배양하였다. Inoculate melan-a cells in 24 well plates at 1 × 10 5 cells per well and for 24 hours at 37 ° C., 10% CO 2 After cultivation in the cell incubator, the roe deer extract was incubated for 3 days at the concentration of 12.5 ㎍ / ㎖, 25 ㎍ / ㎖, 50 ㎍ / ㎖ and 100 ㎍ / ㎖ in each well.

여기에 PBS 2 ㎎/㎖의 농도로 제조한 MTT 용액 200 ㎕를 첨가하고 동일한 배양 조건으로 4 시간을 배양하였다. 200 μl of the MTT solution prepared at a concentration of 2 mg / ml PBS was added thereto, followed by incubation for 4 hours under the same culture conditions.

배양액을 제거하고 각 well당 DMSO 200 ㎕를 가하여 ELISA reader(μQuant, USA)로 570 ㎚에서 흡광도를 측정하였다.The culture solution was removed, and 200 μl of DMSO was added to each well, and the absorbance was measured at 570 nm with an ELISA reader (μQuant, USA).

그 결과, 도 9에서 보는 바와 같이 대조군의 세포 생존률을 100 %로 하였을 때, 노루참나물의 조추출물 농도 중 12.5, 25 ㎍/㎖ 농도에서는 각각 96 %, 97 %로 대조군에 비해 약간 감소하였으나, 50 ㎍/㎖ 농도에서 102 %, 100 ㎍/㎖ 농도에서 107 %로 대조군과 비슷하였다. As a result, as shown in FIG. 9, when the cell viability of the control group was 100%, 12.5 and 25 µg / ml concentrations of crude extracts of the roe deer were 96% and 97%, respectively, slightly decreased compared to the control group. 102% at μg / ml concentration and 107% at 100 μg / ml concentration were similar to the control group.

이처럼 대조군에 비해 약간의 차이가 있으나 유의할만한 변화를 나타내지 않았다.As such, there was a slight difference compared to the control group, but there was no significant change.

<실험예 4> 본 발명의 노루참나물 추출물에 대한 멜라닌 생합성 억제활성 실험Experimental Example 4 Melanin Biosynthesis Inhibitory Activity of the Roe Deer Sprout Extract of the Present Invention

Melan-a 세포주는 마우스 유래의 멜라닌 생성세포로서 멜라닌을 합성한다.Melan-a cell line synthesizes melanin as melanocytes derived from mouse.

본 발명의 노루참나물 조추출물을 12.5㎍/㎖, 25㎍/㎖, 50㎍/㎖ 그리고 100㎍/㎖까지 다양한 농도로 3 일 동안 처리한 후, 멜라닌 생성량을 측정하였다. The crude roe deer extract of the present invention was treated with various concentrations up to 12.5 µg / ml, 25 µg / ml, 50 µg / ml and 100 µg / ml for 3 days, and then the melanin production was measured.

melan-a 세포를 24 well plate에 well당 1×105개의 세포를 접종하고 24 시간동안 37 ℃, 10 % CO2 세포배양기에서 배양한 후 노루참나물 추출물을 각각의 well에 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖의 농도로 3 일간 시료 처리 후 배지를 제거하고 세포를 PBS로 2회 세척하였다. Inoculate melan-a cells in 24 well plates at 1 × 10 5 cells per well and for 24 hours at 37 ° C., 10% CO 2 After culturing in a cell culture medium, the roe deer extracts were sampled at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml in each well for 3 days, and then the medium was removed. Washed twice.

각 well당 500㎕의 1N NaOH를 가하고 56℃에서 30분 용해한 후 ELISA reader (μQuant, USA)로 405 ㎚에서 흡광도를 측정하였다. 500 μl of 1N NaOH was added to each well and dissolved at 56 ° C. for 30 minutes, and then absorbance was measured at 405 nm with an ELISA reader (μQuant, USA).

측정한 결과 대조군인 melazove 30 uM((주) 태평양) 처리시 77 %의 멜라닌 생성 억제활성을 보였으며, 각각 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖ 농도까지 처리시 11 %, 23 %, 42 %, 62 %로 억제활성을 보였다(도10A). As a result of the control, melazove 30 uM (Pacific Pacific) treated group showed 77% melanin inhibitory activity, and treated to 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL, respectively. 11%, 23%, 42%, and 62% of the cells showed inhibitory activity (Fig. 10A).

따라서, 노루참나물 조추출물은 melan-a 세포에서 투여량이 처리 농도가 증가 함에 따라 멜라닌 생성이 억제되는 경향을 보였으며, 통계학적으로 유의성이 있음을 보여주었다. Therefore, the extract of roe deer yam extract showed a tendency to inhibit melanin production as the dose was increased in melan-a cells, and it was statistically significant.

분획물의 멜라닌 억제효과는 에틸아세테이트 분획물이 48 %로 가장 높은 효과를 보였다(도 10B).Melanin inhibitory effect of the fraction showed the highest effect of the ethyl acetate fraction 48% (Fig. 10B).

<실험예 5> 본 발명의 노루참나물 추출물에 대한 세포내 티로시나제 저해활성 실험Experimental Example 5 Experimental Study of Intracellular Tyrosinase Inhibitory Activity of the Roe Deer Fructus Extract of the Present Invention

노루참나물 조추출물 처리 후 최종 멜라닌양이 억제된 것은 멜라닌 합성에 관여하는 효소들의 활성과 관련이 있음을 나타내므로 melan-a 세포에서 티로시나제(tyrosinase) 활성을 측정하였다.Inhibition of the final melanin amount after the crude extract of roe deer sprouts was related to the activity of enzymes involved in melanin synthesis. Therefore, tyrosinase activity was measured in melan-a cells.

melan-a 세포를 24 well plate에 well당 1×105개의 세포를 접종하고 24 시 간동안 37 ℃, 10 % CO2 세포배양기에서 배양한 후 노루참나물 추출물을 각각의 well에 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖의 농도로 처리한 후, well의 세포를 원심분리하여 세포 침전물을 만들고, lysis buffer (0.1 M sodium phosphate buffer, 0.2 mM PMSF, 1% Triton X-100)를 가하였다. Inoculate melan-a cells in a 24 well plate at 1 × 10 5 cells per well, 37 ° C., 10% CO 2 for 24 h. After culturing in a cell incubator, the roe deer sprout extract was treated to each well at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml, and then the cells of the well were centrifuged to obtain cell precipitates. Lysis buffer (0.1 M sodium phosphate buffer, 0.2 mM PMSF, 1% Triton X-100) was added.

얼음에 방치하여 세포를 파괴시키고 원심분리한 후 상층액을 취하여 효소활성측정에 사용하였다. After standing on ice to destroy cells and centrifugation, the supernatant was taken and used for enzyme activity measurement.

각 시료를 반응액에 (12.5 mM L-Dopa, 1.5 mM L-Tyrosine, 67 mM sodium phosphate buffer(pH6.8))넣고 37 ℃에서 1 시간동안 반응시킨 후 ELISA reader를 이용하여 405 nm에서 흡광도를 측정하였다.Each sample was added to the reaction solution (12.5 mM L-Dopa, 1.5 mM L-Tyrosine, 67 mM sodium phosphate buffer (pH6.8)) and reacted at 37 ° C for 1 hour, followed by absorbance at 405 nm using an ELISA reader. Measured.

그 결과, melazove 30 uM((주) 태평양) 처리시 75 %의 티로시나제 억제활성을 보였으며, 노루참나물 조추출물을 12.5 ㎍/㎖, 25 ㎍/㎖, 50 ㎍/㎖ 그리고 100 ㎍/㎖ 농도 처리시 각각 45 %, 59 %, 66 %, 74 %의 억제활성을 보였다(도 11A).As a result, it showed 75% tyrosinase inhibitory activity when treated with melazove 30 uM (Pacific Co., Ltd.), and 12.5 ㎍ / mL, 25 ㎍ / mL, 50 ㎍ / mL and 100 ㎍ / mL concentrations of the roe deer extract were treated. 45%, 59%, 66%, 74% of inhibitory activity, respectively (FIG. 11A).

따라서, 노루참나물 조추출물은 melan-a 세포에서 처리농도가 증가함에 따라 티로시나제 활성도가 억제되는 경향을 보았으며, 통계학적으로 유의성이 있음을 보여 주었다. Therefore, the crude extracts of roe deer were found to have a tendency of inhibiting tyrosinase activity with increasing treatment concentration in melan-a cells and showed statistical significance.

또한, 본 발명의 노루참나물의 추출분획물에 대한 세포 티로시나제 억제효과는 에틸아세테이트 분획물에서 25 ㎍(74 %), 50 ㎍(78 %)의 높은 억제효과를 보였다(도 11B). In addition, the cell tyrosinase inhibitory effect on the extract fraction of the roe deer sprout of the present invention showed a high inhibitory effect of 25 ㎍ (74%), 50 ㎍ (78%) in the ethyl acetate fraction (Fig. 11B).

<실험예 6> 본 발명의 노루참나물 추출물에 대한 항염활성 실험Experimental Example 6 Anti-inflammatory Activity Test of Roe Deer Sprout Extract of the Present Invention

본 발명의 노루참나물 추출물에 대하여 RAW264.7cell에서의 Cell NO 억제효과 및 세포독성저해 효과를 측정하였다.The cell NO inhibitory effect and cytotoxicity inhibitory effect in RAW264.7 cells were measured for the roe deer extract of the present invention.

Raw264.7 cell배양은 DMEM 배지에 10%FBS, 1% 스트렙토마이신/페니실린 (stereptomycine/penicillin)을 첨가하여 세포를 유지하였다.Raw264.7 cell culture was maintained by adding 10% FBS, 1% streptomycin / penicillin (stereptomycine / penicillin) to DMEM medium.

세포농도 1.5×105 cell/㎖를 24 well plate에 seeding 한 후 18 시간 동안 배양하였다. Cell concentration 1.5 × 10 5 cell / ml was seeded in a 24 well plate and incubated for 18 hours.

시료처리는 100 ㎍/㎖과 LPS(lipopolysaccaride) 1 ㎍/㎖를 동시에 처리한 후 24 시간 배양하였다. The sample was incubated for 24 hours after 100 μg / ml and 1 μg / ml of LPS (lipopolysaccaride).

NO생성 측정은 Griess 시약(1% sulfanilamide, 0.1% naphylethylene diamine in 2.5% phosphoric acid) 100 ㎕와 배양액 100 ㎕을 혼합한 후 10 분 동안 반응시켰다. NO production was measured by mixing 100 μl of Griess reagent (1% sulfanilamide, 0.1% naphylethylene diamine in 2.5% phosphoric acid) and 100 μl of the culture solution for 10 minutes.

그리고 540 nm에서 ELISA reader를 이용하여 흡광도를 측정하였다. And absorbance was measured using an ELISA reader at 540 nm.

그 결과, IC50값은 75 ㎍으로서 비교적 높은 억제효과를 보였다.As a result, the IC 50 value was 75 µg, which showed a relatively high inhibitory effect.

또한, MTT assay 결과 100 ㎍/㎖처리까지 세포독성은 없었다. In addition, MTT assay showed no cytotoxicity until 100 ㎍ / ㎖ treatment.

<실험예 7> 본 발명의 노루참나물 추출물에 대한 항산화활성 실험Experimental Example 7 Antioxidant Activity of the Roe Deer Sprout Extract of the Present Invention

1. DPPH 라디칼 소거활성1. DPPH radical scavenging activity

항산화 물질의 가장 특이적인 기작은 유리기와 반응하는 것으로 유리기 소거 작용은 활성라디칼(free radical)에 전자를 공여하여 식물 중의 항산화 효과나 인체에서 노화를 억제하는 척도로 사용된다. The most specific mechanism of antioxidants is the reaction with free radicals. The free radical scavenging action is used as a measure of the antioxidant effect in plants by inhibiting aging in the human body by donating electrons to free radicals.

전자공여능(electron donating ability)측정은 Blosis방법에 의한 DPPH 자유라디칼 소거법에 따라 측정하였다.Electron donating ability was measured by DPPH free radical scavenging method by Blosis method.

먼저 메탄올에 녹인 다양한 농도의 시료를 각각 96 well plate에 100 ㎕씩 분주하고 0.4 mM DPPH용액을 동량으로 첨가하여 실온에서 10분 동안 반응시킨 후 517 ㎚에서 측정하였다. First, 100 μl of various concentrations of samples dissolved in methanol were dispensed into 96 well plates, and 0.4 mM DPPH solution was added in the same amount to react for 10 minutes at room temperature, and then measured at 517 nm.

이때, 대조군으로는 아스코르빈산(ascrbic acid), 트롤록스(trolox)를 사용하였다. At this time, ascorbic acid (ascrbic acid), trolox (trolox) was used as a control.

DPPH 라디칼 소거활성은 아래의 식으로부터 산출하였으며, DPPH의 흡광도가 50 % 시료의 농도(IC50)로 표시하였다. DPPH radical scavenging activity was calculated from the following equation, and the absorbance of DPPH was expressed as the concentration of the 50% sample (IC 50 ).

DPPH radical 소거활성(%)= (A control-Asample)/Acontrol ×100DPPH radical scavenging activity (%) = (A control -A sample ) / A control × 100

Asample = 시료를 첨가한 반응액의 흡광도A sample = absorbance of the reaction solution to which the sample was added

Acontrol = 시료대신 메탄올을 첨가한 반응액 흡광도A control = absorbance of reaction solution with methanol instead of sample

그 결과, 상용화된 아스코르브산(ascorbic acid)의 IC50은 8.23 ㎍으로 높은 활성라디칼 억제효과를 보였다. As a result, IC 50 of commercialized ascorbic acid showed 8.23 ㎍ with high active radical inhibition effect.

노루참나물 조추출물의 IC50은 227.32 ㎍으로 낮은 효과를 보였으나, 노루참 나물 에틸아세테이트 분획물은 IC50이 79.76 ㎍, 부탄올 분획물은 IC50이 161.33 ㎍/㎖)층은 조추출물보다 2 배 이상 활성산소 억제 효과를 보였다. And showed an IC 50 is low effect 227.32 ㎍ of roe chamnamul crude extract, deer true herbs ethyl acetate fraction IC 50 is 79.76 ㎍, butanol fraction is the IC 50 layer 161.33 ㎍ / ㎖) is twice as active than the crude extract It showed oxygen suppression effect.

2. Nitirc oxide 소거활성 측정2. Measurement of Nitirc oxide scavenging activity

질소화합물은 세포독성이 강하며 다량의 NO가 생성되면 아질산화(nitrozation), 니트로화(nitration)와 같은 간접적인 효과 및 산화반응을 야기하여 유해효과를 가져온다. Nitrogen compounds are highly cytotoxic and when a large amount of NO is produced, they cause indirect effects such as nitrozation and nitration and oxidative reactions, resulting in harmful effects.

본 실험에서는 qucertin을 대조군으로 하여 본 발명의 노루참나물 추출물의 nitric oxide 생성저해 활성을 측정하였다.In this experiment, the inhibitory activity of nitric oxide production of the roe deer extract of the present invention was measured using qucertin as a control.

nitric oxide을 생성하는 니트로프루시드나트륨(sodium nitroprusside, SNP)을 사용하여 아질산염의 양을 측정하는 방법으로 NO소거 활성을 측정하였다. The NO scavenging activity was measured by measuring the amount of nitrite using sodium nitroprusside (SNP) that produces nitric oxide.

10 mM SNP용액에 200 ㎕에 시료를 농도별로 첨가하고 25 ℃에서 3 시간 동안 반응시킨후 동량의 Griess reagent(2.5% phophoric acid, 1% sulfanilamide, 0.1% naphylethylendiamine)를 첨가하여 실온에서 10 분간 반응하여 540 nm에서 흡광도를 측정하였다. Samples were added to 200 μl of 10 mM SNP solution by concentration, and reacted at 25 ° C. for 3 hours. Then, the same amount of Griess reagent (2.5% phophoric acid, 1% sulfanilamide, 0.1% naphylethylendiamine) was added for 10 minutes Absorbance was measured at 540 nm.

Nitric oxide 소거활성은 흡광도가 50 %감소할 때 나타나는 시료의 농도 (IC50)로 표시하였다. Nitric oxide scavenging activity was expressed as the concentration of the sample (IC 50 ) that appears when the absorbance was reduced by 50%.

그 결과, 노루참나물 조추출물과 물분획, 에틸아세테이트 분획물, 부탄올 분획물에서는 저해율이 매우 낮았으나, 헥산 분획물에서는 IC50이 123.53으로 억제효 과를 보였다.As a result, the inhibition rate was very low in the crude extract, water fraction, ethyl acetate fraction, butanol fraction, but the IC 50 was 123.53 in the hexane fraction.

3. 잔틴 산화효소 억제 및 과산화물 소거활성 측정3. Xanthine Oxidase Inhibition and Peroxide Scavenging Activity

xantihine/xanthine oxidase에 의한 요산(uric acid) 생성은 290 nm에서 증가된 흡광도에 의해 측정하였다. The production of uric acid by xantihine / xanthine oxidase was measured by increased absorbance at 290 nm.

과산화물(superoxide)의 양은 니트로블루 테트라졸리움(nitroblue tetrazolium, NBT)환원방법에 의해 측정하였다. The amount of superoxide was measured by the nitroblue tetrazolium (NBT) reduction method.

반응액(0.5mM xanthine, 200 mM phosphate buffer(pH7.5) 1mM EDTA)에 50mU/ml 크산틴옥사이드(xanthine oxidase)를 첨가하여 요산(uric acid)생성을 유도하였다. The reaction solution (0.5mM xanthine, 200 mM phosphate buffer (pH7.5) 1mM EDTA) was added to 50mU / ml xanthine oxidase to induce the production of uric acid (uric acid).

과산화물(superoxide) 소거활성은 위 반응액에 0.5mM NBT를 첨가하여 반응하였다. Superoxide scavenging activity was reacted by adding 0.5mM NBT to the reaction solution.

잔틴 산화효소(Xanthine oxidase) 억제 및 과산화물(superoxide) 소거활성은 각각 생성된 요산(uric acid)과 과산화물의 흡광도가 50 %이상 감소할 때 나타나는 시료의 농도(IC50)로 표시하였다.Xanthine oxidase inhibition and superoxide scavenging activity were expressed as the concentration of the sample (IC 50 ) which appeared when the absorbance of the produced uric acid and peroxide decreased by more than 50%.

그 결과, 노루참나물 에탄올 조추출물을 포함한 분획에서의 잔틴 산화효소(xanthine oxidase) 억제효과는 매우 낮았다.As a result, the inhibitory effect of xanthine oxidase was very low in the fraction containing the ethanol extract of roe deer ethanol.

그러나, 과산화물 소거활성은 노루참나물 조추출물은 IC50가 30.79 ㎍/㎖, 에틸아세테이트 분획물은 IC50이 8.97 ㎍/㎖, 부탄올 분획물의 IC50은 14.03 ㎍/㎖, 헥산분획물의 IC50은 151.33 ㎍/㎖, 물 분획물은 IC50이 >400 ㎍/㎖으로서 에틸아세테이트 분획물에서 높은 억제효과를 나타내었다. However, peroxide scavenging activity deer chamnamul crude extract of IC 50 is 30.79 ㎍ / ㎖, ethyl acetate fraction IC 50 is 8.97 ㎍ / ㎖, IC 50 of the butanol fraction was 14.03 ㎍ / ㎖, IC 50 of the hexane fraction was 151.33 ㎍ / Ml, water fraction showed a high inhibitory effect on the ethyl acetate fraction as IC 50 > 400 μg / ml.

<표 3> 노루참나물 추출물 및 추출분획물에 대한 항산화 활성 실험 결과<Table 3> Antioxidant Activity Test Results of Roe Deer Sprout Extract and Extract Fraction

구 분division DPPH(IC50)DPPH (IC 50 ) NO(IC50)NO (IC 50 ) 잔틴산화효소 억제활성(IC50)Xanthine oxidase inhibitory activity (IC 50 ) 과산화물 소거활성(IC50)Peroxide Scavenging Activity (IC 50 ) 조추출물Crude extract 227227 >400> 400 -- 30.7930.79 헥산분획물Hexane fraction >400> 400 123.52123.52 219219 151.33151.33 에틸아세테이트 분획물Ethyl acetate fraction 79.779.7 >400> 400 125.78125.78 8.978.97 부탄올분획물Butanol fraction 161.33161.33 >400> 400 187.93187.93 14.0314.03 물 분획물Water fraction >400> 400 13.5313.53 >400> 400 >400> 400 대조구Control Asco 8.23Asco 8.23 Qucertin 13.53Qucertin 13.53 -- Allo 0.028Allo 0.028

본 발명에 의해 미백 활성이 뛰어나고, 항산화 및 항염활성이 뛰어난 노루참나물 추출물이 제공된다.According to the present invention, a roe deer extract having excellent whitening activity and excellent antioxidant and anti-inflammatory activity is provided.

또한, 본 발명의 노루참나물 추출물을 유효성분으로 포함하는 조성물이 제공된다.In addition, there is provided a composition comprising the roe deer extract of the present invention as an active ingredient.

또한, 본 발명의 노루참나물 추출물로부터 분리한 카페익산 메틸 에스테르가 제공되고, 이 분리된 물질을 유효성분으로 포함하는 조성물이 제공된다.In addition, there is provided a caffeic acid methyl ester separated from the roe deer extract of the present invention, a composition comprising the separated substance as an active ingredient.

Claims (7)

노루참나물(Pimpinella komarovii)로 부터 추출한 것을 특징으로 한 미백활성을 나타내는 노루참나물 추출물.Roe deer roe extract, which has whitening activity, characterized in that it is extracted from Pimpinella komarovii . 제1항에 있어서,The method of claim 1, 노루참나물 추출물은, 노루참나물을 세척한 후 3일 동안 40 ℃로 열풍건조한 다음 분쇄하고, 이 노루참나물 분쇄물을 70 % 에탄올에 침적시키고 2 ~ 3 일동안 실온에서 교반하여 침출한 다음, 여과기로 여과하여 조추출물을 제조하고, 이 조추출물을 극성이 다른 용매인 n- 헥산, 에틸아세테이트, 부탄올을 이용해 순차적으로 분획하여 추출분획물을 제조하는 것이 특징인, The roe deer extract is hot-dried at 40 ° C. for 3 days after washing the roe deer sprout, and then pulverized. The roe deer crushed powder is immersed in 70% ethanol and stirred for 2 to 3 days at room temperature, followed by filtration. The crude extract is prepared by filtration, and the crude extract is sequentially fractionated using n-hexane, ethyl acetate, butanol, which are solvents having different polarities, to prepare an extract fraction. 미백활성을 나타내는 노루참나물 추출물.A roe deer extract showing whitening activity. 제1항에 있어서,The method of claim 1, 노루참나물 추출물은 멜라닌 생합성 억제활성 또는 티로시나제 저해활성을 나타내는 것이 특징인,Roe deer bran extract is characterized by showing melanin biosynthesis inhibitory activity or tyrosinase inhibitory activity, 미백활성을 나타내는 노루참나물 추출물.A roe deer extract showing whitening activity. 노루참나물(Pimpinella komarovii) 추출물을 유효성분으로 포함하는 미백용 조성물.Roe deer Pimpinella komarovii ) Whitening composition comprising the extract as an active ingredient. 노루참나물(Pimpinella komarovii) 추출물을 유효성분으로 포함하는 염증성 질환의 개선 및 예방용 조성물.Roe deer Pimpinella komarovii ) A composition for improving and preventing inflammatory diseases comprising an extract as an active ingredient. 노루참나물(Pimpinella komarovii) 추출물을 유효성분으로 포함하는 항산화용 조성물.Roe deer Pimpinella komarovii ) Antioxidant composition comprising the extract as an active ingredient. 노루참나물(Pimpinella komarovii) 추출물로부터 분리된 카페익산 메틸 에스테르(Caffeic acid methyl ester)를 유효성분으로 포함하는 미백용 조성물.Roe deer Pimpinella komarovii ) Whitening composition comprising caffeic acid methyl ester isolated from the extract as an active ingredient.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR960022431A (en) * 1994-12-28 1996-07-18 성재갑 How to Extract Caffeic Acid from Buttercup
US20030099726A1 (en) 1996-02-12 2003-05-29 Squires Meryl J. Antimicrobial treatment for herpes simplex virus and other infectious diseases
KR20050052839A (en) * 2003-12-01 2005-06-07 주식회사 태평양 Skin compositions for exteral application, containing plant extracts
KR20070022178A (en) * 2005-08-18 2007-02-26 한불화장품주식회사 Cosmetic composition containing extract of pimpinella brachycarpa

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR960022431A (en) * 1994-12-28 1996-07-18 성재갑 How to Extract Caffeic Acid from Buttercup
US20030099726A1 (en) 1996-02-12 2003-05-29 Squires Meryl J. Antimicrobial treatment for herpes simplex virus and other infectious diseases
KR20050052839A (en) * 2003-12-01 2005-06-07 주식회사 태평양 Skin compositions for exteral application, containing plant extracts
KR20070022178A (en) * 2005-08-18 2007-02-26 한불화장품주식회사 Cosmetic composition containing extract of pimpinella brachycarpa

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