KR20050081810A - Angelica keiskei preparation for antioxidative function containing Inteolin 7-O-β-D-glucopyranoside, quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-galactopyranoside, quercetin 3-O-α-D-arabinopyranoside, kaempferol 3-O-α-D-arabinopyranoside, luteolin 3-O-β-D-rhamnopyranosyl(1→6)-7-O-α-L-glucopyranoside and preparation method thereof - Google Patents

Angelica keiskei preparation for antioxidative function containing Inteolin 7-O-β-D-glucopyranoside, quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-galactopyranoside, quercetin 3-O-α-D-arabinopyranoside, kaempferol 3-O-α-D-arabinopyranoside, luteolin 3-O-β-D-rhamnopyranosyl(1→6)-7-O-α-L-glucopyranoside and preparation method thereof Download PDF

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KR20050081810A
KR20050081810A KR1020040011177A KR20040011177A KR20050081810A KR 20050081810 A KR20050081810 A KR 20050081810A KR 1020040011177 A KR1020040011177 A KR 1020040011177A KR 20040011177 A KR20040011177 A KR 20040011177A KR 20050081810 A KR20050081810 A KR 20050081810A
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glucopyranoside
quercetin
luteolin
arabinopyranoside
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박근형
김소중
조정용
문제학
위지향
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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    • A61K2236/55—Liquid-liquid separation; Phase separation

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Abstract

본 발명은 천연항산화물질을 포함한 항산화 기능을 갖는 신선초조제물 및 제조에 관한 것이다. 보다 상세하게는 신선초를 열수로 추출한 추출물을 용매분획하여 얻어진 에칠아세테이트가용중성획분을 세파덱스 LH-20 컬럼 크로마토그라피 및 HPLC를 의해 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드를 분리하고 이들 물질의 구조결정은 MS, NMR 분석에 의해 이루어졌다. 이들 물질들은 DPPH 자유라디칼소거에 대한 항산화 활성을 갖고 있음이 검정되어, 이들 천연항산화물질을 포함한 항산화 기능을 갖는 신선초조제물은 산화에 의한 질병예방 및 노화억제효력을 갖는 기능성소재로서의 용도를 제공한다.The present invention relates to fresh preparations and preparations having an antioxidant function, including natural antioxidants. More specifically, the ethyl acetate soluble neutral fraction obtained by solvent fractionation of the extract extracted from fresh vinegar with hot water was subjected to luteolin 7- O- β-D-glucopyranoside and quesertin by Sephadex LH-20 column chromatography and HPLC. 3- O- β-D-galactopyranoside, quercetin 3- O- β-D-glucopyranoside, quercetin 3- O- α-D-arabinopyranoside, campello 3- O -α-D-arabinopyranoside, luteolin 3- O- β-D-ramnopyranosyl (1 → 6) -7- O- α-L-glucopyranoside, isolated and structural determination of these materials Was carried out by MS, NMR analysis. These substances have been tested to have antioxidant activity against DPPH free radical scavenging, and fresh herbal preparations with antioxidant functions, including these natural antioxidants, provide their use as functional materials with anti-oxidative disease prevention and anti-aging effects. .

Description

항산화 활성을 갖는 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드 등의 천연 항산화물질을 포함하여 항산화기능을 갖는 신선초조제물 및 이의제조방법 {Angelica keiskei preparation for antioxidative function containing Inteolin 7-O-β-D-glucopyranoside, quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-galactopyranoside, quercetin 3-O-α-D-arabinopyranoside, kaempferol 3-O-α-D-arabinopyranoside, luteolin 3-O-β-D-rhamnopyranosyl(1→6)-7-O-α-L-glucopyranoside and preparation method thereof}Luteolin 7-O-β-D-glucopyranoside with antioxidant activity, quercetin 3-O-β-D-galactopyranoside, quercetin 3-O-β-D-glucopyranoside, Quercetin 3-O-α-D-arabinopyranoside, Campello 3-O-α-D-arabinopyranoside, luteolin 3-O-β-D-ramnopyranosyl (1 → 6) Fresh kelp preparation with antioxidant function, including natural antioxidants such as -7-O-α-L-glucopyranoside, and method for preparing the same , quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-galactopyranoside, quercetin 3-O-α-D-arabinopyranoside, kaempferol 3-O-α-D-arabinopyranoside, luteolin 3-O- β-D-rhamnopyranosyl (1 → 6) -7-O-α-L-glucopyranoside and preparation method

본 발명은 천연항산화물질을 포함한 항산화 기능을 갖는 신선초조제물 및 제조에 관한 것이다. 보다 상세하게는 신선초지상부를 동결건조하여 분말화한 다음 열수로 추출한후 얻어진 추출물을 용매분획하여 얻은 에칠아세테이트가용중성획분을 세파덱스 LH-20 컬럼 크로마토그라피 및 HPLC를 의해 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드를 분리하고 이들 물질의 구조결정은 MS, NMR 분석에 의해 이루어졌다. 이들 물질들은 DPPH 자유라디칼소거에 대한 항산화 활성을 갖고 있음이 검정되어, 이들 천연항산화물질을 포함한 신선초조제물은 항산화 기능을 갖는 기능성소재 등의 용도를 제공한다.The present invention relates to fresh preparations and preparations having an antioxidant function, including natural antioxidants. More specifically, lyophilized fresh ground portion was pulverized and extracted with hot water, and then the ethyl acetate soluble neutral fraction obtained by solvent fractionation of the extracted extract was purified by luteolin 7- O- β by Sephadex LH-20 column chromatography and HPLC. -D-glucopyranoside, quercetin 3- O- β-D-galactopyranoside, quercetin 3- O- β-D-glucopyranoside, quercetin 3- O- α-D-ara Vinopyranoside, Campello 3- O- α-D-Arabinopyranoside, Luteolin 3- O- β-D-Ranmopyranosyl (1 → 6) -7- O- α-L-Glucopi Llanoside was separated and the structure of these materials was determined by MS, NMR analysis. These substances have been assayed to have antioxidant activity against DPPH free radical scavenging, and fresh herbal preparations containing these natural antioxidants provide applications such as functional materials with antioxidant functions.

인간을 비롯한 모든 생물체들은 공기중의 산소를 이용하여 생명유지에 필요한 에너지를 발생하지만, 각종 물리·화학적 요인 등에 의해 활성산소의 생성이 증가되면 생체내 산화적 손상을 받게 되어 직접 또는 간접적으로 생체 장애를 일으키는 원인이 되고 있다. 따라서 이를 방어하기 위한 기능성을 갖는 식품은 질병의 예방이나 노화억제 등 인간의 건강을 유지하는데 중요한 역할을 하며, 적절한 식품을 통해 이들 질병의 예방과 치유가 가능하다는 인식이 확산됨에 따라 예방의학적 측면에서 식품의 중요성이 대두되고 있다.All living organisms, including humans, use the oxygen in the air to generate the energy needed to sustain life, but when the production of free radicals increases due to various physical and chemical factors, oxidative damage is caused in vivo. It is causing. Therefore, foods with a function to defend them play an important role in maintaining human health such as disease prevention and aging control, and in the preventive medical aspect, as the awareness that proper foods can prevent and cure these diseases is spreading. The importance of food is on the rise.

신선초는 미나리과에 속하는 아열대성 다년생 초본으로, 명일엽, 신립초, 선상초 등으로 불리고 고혈압, 당뇨, 간장병, 신경통, 동맥경화 등 성인병 예방 목적으로 차, 생즙, 분말 등의 형태로 이용되고 있다 [임웅규등, 신선초, 광일문화사, 13, 1991] . 신선초지상부에 함유된 기능성 물질에 관한 선행연구로는, 루테올린 7-글루코사이드, 루테올린 3-루티노사이드, 그리고 퀘서틴 3-갈락토사이드 [박종철 등, 미나리과 식용식물의 생리활성과 이들의 기능성 플라보노이드 화합물, 식품산업과 영양, 7, 2, 30-34, 2002] 가 메탄올추출물에서 발견되었고 이중 루테올린 7-글루코사이드와 퀘서틴 3-갈락토사이드는 콜레스테롤 합성 저해작용 [Park, J.R. et al., Screening and characterization of anticholesterogenic substances from edible plant extracts. Han'guk Sikp'um Yongyang Kwahak Hoechi, 26, 236-241, 1997] 이 보고된 바 있으나, 본 발명에서 발견한 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드 등의 천연 항산화 물질이 신선초지상부에 함유되어 있음이 보고된 바 없다.Sinseoncho is a subtropical perennial herb belonging to the Araaceae family, and it is called Myung Il-yeop, Sincho-cho, and Seoncho-cho, and is used in the form of tea, juice, powder, etc. for the purpose of preventing adult diseases such as hypertension, diabetes, liver disease, neuralgia, and arteriosclerosis. Et al., Sinseoncho, Kwangil Cultural History, 13, 1991]. Prior studies on functional substances contained in fresh grassland include luteolin 7-glucoside, luteolin 3-lutinoside, and quercetin 3-galactoside [Park Jong-Chul et al. Flavonoid Compounds, Food Industry and Nutrition, 7, 2, 30-34, 2002] were found in methanol extracts, of which luteolin 7-glucoside and quercetin 3-galactoside inhibited cholesterol synthesis [Park, JR et al . , Screening and characterization of anticholesterogenic substances from edible plant extracts. Han'guk Sikp'um Yongyang Kwahak Hoechi, 26, 236-241, 1997] , but the bar, a Quebec luteolin 7- O -β-D- glucopyranoside, seotin found in the present invention 3-looking O - β-D-galactopyranoside, quercetin 3- O- β-D-glucopyranoside, quercetin 3- O- α-D-arabinopyranoside, campello 3- O- α-D Natural antioxidants such as arabinopyranoside and luteolin 3- O- β-D-ramnopyranosyl (1 → 6) -7- O- α-L-glucopyranoside are contained in fresh grassland This has not been reported.

신선초지상부에 항산화 활성을 갖는 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드 등의 천연 항산화 활성물질을 포함한 항산화 기능을 갖는 신선초조제물에 천연항산화 활성물질이 존재함을 확인하고, 그 항산화 활성 원인물질을 발견하였으며, 아울러 발견된 물질들의 항산화 활성을 검정하여, 이들 천연항산화물질을 포함하는 신선초조제물을 항산화기능을 갖는 소재로 제공하고자 한다.Having antioxidant activity in the above-ground Angelica keiskei luteolin 7- O -β-D- glucopyranoside, Quebec seotin 3- O -β-D- galacto-pyrano seed, Quebec seotin 3- O -β-D- gluconic nose Llano seed, Quebec seotin 3- O -α-D- arabino pyrano seed, fellow cam 3- O -α-D- arabino pyrano seed, luteolin 3- O -β-D- ramno pyrazol nosil (1 → 6) Confirm that natural antioxidant active substances exist in fresh herb preparations with antioxidant function, including natural antioxidant active substances such as 7- O- α-L-glucopyranoside, and find the cause of the antioxidant activity. In addition, by examining the antioxidant activity of the substances found, it is intended to provide a fresh preparation containing these natural antioxidants as a material having an antioxidant function.

본 발명은 신선초지상부를 동결건조시킨 분말로부터 세파덱스 LH-20 및 HPLC 등에 의해 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드를 각각 분리하였으며, 이들 물질의 구조는 MS, NMR 분석에 의해 결정되었다. 또한 이들 천연항산화물질이 DPPH 라디칼 소거에 대한 항산화 활성을 검정하여 제시하였다.The present invention relates to luteolin 7- O- β-D-glucopyranoside, quercetin 3- O- β-D-galactopyranoside from Sepadex LH-20, HPLC and the like from the lyophilized powder , Quebec seotin 3- O -β-D- glucopyranoside, Quebec seotin 3- O -α-D- arabino pyrano seed, fellow cam 3- O -α-D- arabino pyrano seed, De ruthenate 3- O- β-D-ramnopyranosyl (1 → 6) -7- O- α-L-glucopyranoside was isolated, respectively, and the structure of these materials was determined by MS, NMR analysis. In addition, these natural antioxidants were shown by assaying the antioxidant activity against DPPH radical scavenging.

이하, 하기 실시예에 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail in the following examples.

실시예 1 : 신선초조제물의 제조 및 조제물의 항산화 활성Example 1 Preparation of Fresh Herb Preparations and Antioxidant Activities of the Preparations

신선초(Angelica keiskei Koidz)는 신선초 지상부를 동결건조하여 분말화한 것을 시료로 사용하였다. 신선초추출물의 추출 및 용매분획은 다음과 같이 하였다. 즉, 신선초 건조분말(2.5 kg)을 열수(90℃, 30분)로 추출한 후, 여과하여 얻어진 상등액을 5% NaHCO3을 이용하여 pH 8.0으로 조정한 다음, 에칠아세테이트(EtOAc)로 분배하여 에칠아세테이트가용중성획분(EtOAc-soluble neutral fraction)과 수상획분(Aqeous acidic fraction)으로 분획하였다. 이 수상 획분에 1.0 N HCl을 가하여 pH 3.0으로 조절한 후, 다시 에칠아세테이트로 분배하여 에칠아세테이트가용산성획분(EtOAc-soluble acidic fraction)과 수용성 획분(Aqeous fraction)으로 분획하였다. 신선초지상부열수추출물의 용매분획에 의해 에칠아세테이트가용산성획분(12.5 g)과 에칠아세테이트가용중성획분(2.4 g)을 각각 얻었다. Angelica keiskei Koidz used lyophilized ground powder of fresh vinegar as a sample. Extraction and solvent fraction of fresh vinegar extract were as follows. That is, after extracting the fresh vinegar dry powder (2.5 kg) with hot water (90 ℃, 30 minutes), the supernatant obtained by filtration was adjusted to pH 8.0 using 5% NaHCO 3 , and then partitioned with ethyl acetate (EtOAc) It was partitioned into acetate soluble neutral fraction (EtOAc-soluble neutral fraction) and aqueous fraction (Aqeous acidic fraction). 1.0 N HCl was added to the aqueous phase to adjust the pH to 3.0, and then partitioned into ethyl acetate, which was then partitioned into an ethyl acetate soluble acidic fraction (EtOAc-soluble acidic fraction) and an aqueous fraction (Aqeous fraction). Solvent fractions of fresh superground hydrothermal extracts yielded ethyl acetate soluble acid fraction (12.5 g) and ethyl acetate soluble neutral fraction (2.4 g), respectively.

얻어진 이들 획분의 항산화 활성측정은, DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma, St. Louis, MO, USA) 라디칼 소거활성 검정법으로 측정하였다. 즉, Abe 등의 방법 [Abe, N. et al., Studies of the 1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2-pyrone compound, Biosci. Biotech. Biochem., 64, 306-333, 2000] 에 의해 DPPH의 에탄올용액(100 μM) 2700 μL에 추출물이 함유된 시료용액 300 μL를 시험관에 넣고, 볼텍스 혼합기(vortex mixer)로 가볍게 혼합한 다음, 암소에서 30분간 반응시킨 후, 517 nm에서 흡광도를 측정하였다. 또한, 비교구로 천연항산화제인 알파 토코페롤을 이용하였으며, DPPH 라디칼이 50% 소거되어진 농도를 SC50 (50% scavenging concentration)으로 나타냈다. 그 결과, 50%의 DPPH 라디칼 소거 농도(SC50)는 에칠아세테이트가용산성획분이 21 μg/mL, 에칠아세테이트가용중성획분이 18 μg/mL, 그리고 알파토코페롤은 8.0 μg/mL으로 측정되었다. 열수추출물의 에칠아세테이트가용획분들은 모두 항산화 활성을 보였으며, 이중 에칠아세테이트가용중성획분이 에칠아세테이트가용산성획분보다 강한 활성을 보였다. 따라서 신선초조제물은 항산화 활성을 갖고 있음이 확인되었으며, 이중 보다 높은 활성을 나타낸 에칠아세테이트가용중성획분에 함유된 항산화 활성물질의 분리를 시도하였다.The antioxidant activity of these obtained fractions was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma, St. Louis, MO, USA) radical scavenging activity assay. That is, Abe et al . [Abe, N. et al ., Studies of the 1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2-pyrone compound, Biosci. Biotech. Biochem., 64, 306-333, 2000] 300 μL of the sample solution containing the extract in 2700 μL of the ethanol solution (100 μM) of DPPH was added to the test tube, and gently mixed with a vortex mixer, followed by After reacting for 30 minutes at, the absorbance at 517 nm was measured. In addition, as a control, a natural antioxidant alpha tocopherol was used, and the concentration of 50% scavenging concentration of DPPH radicals was expressed as SC 50 (50% scavenging concentration). As a result, the DPPH radical scavenging concentration (SC 50 ) of 50% was determined to be 21 μg / mL of ethyl acetate soluble acidic fraction, 18 μg / mL of ethyl acetate soluble neutral fraction, and 8.0 μg / mL of alphatocopherol. All of the ethyl acetate soluble fractions of hot water extract showed antioxidant activity, of which the ethyl acetate soluble neutral fraction showed stronger activity than the ethyl acetate soluble acid fraction. Therefore, fresh herbal preparations were found to have antioxidant activity. Among them, an attempt was made to separate the antioxidant active material contained in the ethyl acetate soluble neutral fraction showing higher activity.

이하 실시예 2에서 신선초조제물에 함유된 천연항산화물질의 분리 및 구조해석에 자세히 설명한다.In the following Example 2 will be described in detail in the separation and structural analysis of the natural antioxidants contained in the fresh herb preparation.

실시예 2 : 신선초조제물에 함유된 천연항산화 활성물질의 분리 및 구조해석Example 2 Isolation and Structural Analysis of Natural Antioxidant Active Substances Contained in Fresh Herb Preparations

1) 활성물질의 분리1) Isolation of Active Material

실시예 1에서 제시한 항산화 활성을 갖는 에칠아세테이트가용중성획분(2.4 g)을 메탄올-물 용매계로, 메탄올 함량을 10%씩 증가시키는 용출방법으로 세파덱스 LH-20 컬럼 크로마토그라피에 의해 용출 및 분획하여 DPPH-TLC법에 의한 항산화 활성을 검정한 결과, 50과 70% MeOH의 각 획분에서 항산화 활성이 나타났다. 이중 70% MeOH 용출획분(472 mg)을 마이크로본다팍 컬럼에 의한 HPLC를 실시하여 항산화 활성을 갖는 화합물로 활성물질 1 (15.9 mg, 황색분말), 활성물질 2·3 (156.8 mg, 황색분말), 활성물질 4 (131.4 mg, 황색분말) 및 활성물질 5 (21.8 mg, 황색분말)를 각각 분리하였다. 또한 세파덱스 LH-20 컬럼 크로마토그라피에 의해 얻어진 50% MeOH 용출획분(221.8 mg)을 마이크로본다팍 컬럼에 의한 HPLC를 실시하여 항산화 활성을 갖는 화합물로 활성물질 6 (22.7 mg, 황색분말)을 분리하였다.Elution and fractionation of the ethyl acetate soluble neutral fraction (2.4 g) having the antioxidant activity shown in Example 1 in a methanol-water solvent system was eluted with Sephadex LH-20 column chromatography in an elution method to increase the methanol content by 10%. As a result of assaying antioxidant activity by DPPH-TLC method, antioxidant activity was observed in each fraction of 50 and 70% MeOH. Among them, 70% MeOH eluted fraction (472 mg) was subjected to HPLC using a microbondapak column to have antioxidant activity. Active substance 1 (15.9 mg, yellow powder), active substance 2 · 3 (156.8 mg, yellow powder) Active substance 4 (131.4 mg, yellow powder) and active substance 5 (21.8 mg, yellow powder) were separated. In addition, 50% MeOH eluted fraction (221.8 mg) obtained by Sephadex LH-20 column chromatography was subjected to HPLC using a microbondapak column to separate active substance 6 (22.7 mg, yellow powder) into a compound having antioxidant activity. It was.

2) 활성물질 1(루테올린 7-O-β-D-글루코피라노시드)의 구조해석2) Structural Analysis of Active Substance 1 (Luteolin 7- O- β-D-Glucopyranoside)

활성물질 1을 구조해석하기 위하여 질량분석기(FAB-MS positive, JMS-HX/HX110A Tandem Mass Spectrometer, Jeol, Japan)로 분석을 행한 결과, m/z 449 [M+H]+가 검출되어 이의 분자량이 448임을 알 수 있었다. 기기(UnitINOVA 500)에 의해 분석된 1H-NMR (500 MHz, pyridine-d 5 )의 측정값은 δ 6.94 (1H, s, H-3), 6.86 (1H, d, J = 1.5 Hz, H-6), 7.01 (1H, d, J = 1.5 Hz, H-8), 7.91 (1H, d, J = 1.5 Hz, H-2'), 7.30 (1H, d, J = 8.5 Hz, H-5'), 7.53 (1H, dd, J = 1.5, 8.5 Hz, H-6'), 5.83 (1H, d, J = 7.5 Hz, H-1"), 4.60-3.33 (H-2"∼H-6")이며, 13C-NMR (125 MHz, pyridine-d 5 )에 의해 분석된 측정값은 δ165.80 (C-2), 104.59 (C-3), 183.32 (C-4), 163.06 (C-5), 101.10 (C-6), 164.46 (C-7), 95.80 (C-8), 158.36 (C-9), 107.07 (C-10), 123.18 (C-1'), 115.18 (C-2'), 148.30 (C-3'), 152.41 (C-4'), 117.36 (C-5'), 120.16 (C-6'), 102.28 (C-1"), 75.30 (C-2"), 78.63 (C-3"), 71.64 (C-4"), 79.71 (C-5"), 62.85 (C-6")이었다. 또한 HMBC 분석에서 당의 1위(H-1")와 루테올린의 탄소 7위와 크로스피크를 보여 결합위치를 알 수 있었다. 이상의 MS 및 NMR등의 기기분석결과로부터 이 물질의 구조를 하기와 같이 결정하였다.To analyze the active substance 1 , a mass spectrometer (FAB-MS positive, JMS-HX / HX110A Tandem Mass Spectrometer, Jeol, Japan) showed that m / z 449 [M + H] + was detected. It was found that this is 448. Measurements of 1 H-NMR (500 MHz, pyridine- d 5 ) analyzed by the instrument ( Unit INOVA 500) were δ 6.94 (1H, s, H-3), 6.86 (1H, d, J = 1.5 Hz, H-6), 7.01 (1H, d, J = 1.5 Hz, H-8), 7.91 (1H, d, J = 1.5 Hz, H-2 '), 7.30 (1H, d, J = 8.5 Hz, H -5 '), 7.53 (1H, dd, J = 1.5, 8.5 Hz, H-6'), 5.83 (1H, d, J = 7.5 Hz, H-1 "), 4.60-3.33 (H-2" to H-6 ″), and the measured values analyzed by 13 C-NMR (125 MHz, pyridine- d 5 ) are δ165.80 (C-2), 104.59 (C-3), 183.32 (C-4), 163.06 (C-5), 101.10 (C-6), 164.46 (C-7), 95.80 (C-8), 158.36 (C-9), 107.07 (C-10), 123.18 (C-1 '), 115.18 (C-2 '), 148.30 (C-3'), 152.41 (C-4 '), 117.36 (C-5'), 120.16 (C-6 '), 102.28 (C-1 "), 75.30 ( C-2 "), 78.63 (C-3"), 71.64 (C-4 "), 79.71 (C-5"), 62.85 (C-6 "). Also ranked first in sugar (H-1) in HMBC analysis. ") And luteolin's carbon 7th position and cross peak showed the bond location. From the results of instrumental analysis such as MS and NMR, the structure of this material was determined as follows.

3) 활성물질 2(퀘서틴 3-O-β-D-갈락토피라노시드)의 구조해석3) Structural Analysis of Active Substance 2 (Quercetin 3- O- β-D-galactopyranoside)

활성물질 2를 구조해석하기 위하여 질량분석기(FAB-MS positive, Platform 2)로 분석을 행한 결과, m/z 465 [M+H]+ 및 m/z 487 [M+Na]+이 검출되어 분자량이 464임을 알 수 있었다. 한편 1H- 및 13C-NMR분석에 의하면 대단히 유사한 경향의 시그날들이 약 3:1의 존재비로 검출되어 2종의 화합물이 혼합된 상태임을 알 수 있었다. 그중 보다 존재비가 높은 화합물을 활성물질 2, 그리고 보다 낮은 존재비의 화합물을 활성물질 3으로 명명하였다. 활성물질 2의 구조해석에 대해서는 하기에 기술하며, 활성물질 3은 4)의 구조해석에서 기술한다.To analyze the active substance 2 by mass spectrometry (FAB-MS positive, Platform 2), m / z 465 [M + H] + and m / z 487 [M + Na] + were detected. This was found to be 464. On the other hand, 1 H- and 13 C-NMR analysis showed that signals with a very similar tendency were detected in an abundance ratio of about 3: 1, indicating that two compounds were mixed. Among them, the higher abundance compound was designated as active material 2 , and the lower abundance compound as active material 3. The structural analysis of the active substance 2 is described below, and the active substance 3 is described in the structural analysis of 4).

활성물질 2는 기기(UnitINOVA 500)에 의해 분석된 1H-NMR (500 MHz, CD3 OD)의 측정값은 δ6.16 (1H, d, J = 2.0 Hz, H-6), 6.37 (1H, d, J = 2.0 Hz, H-8), 7.84 (1H, d, J = 2.0 Hz, H-2'), 6.86 (1H, d, J = 8.5 Hz, H-5'), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 5.12 (1H, d, J = 8.0 Hz, H-1"), 4.55-3.33 (H-2"∼H-6")이며, 13C-NMR (125 MHz, CD3OD)에 의해 분석된 측정값은 δ 158.94 (C-2), 135.91 (C-3), 179.90 (C-4), 163.18 (C-5), 100.20 (C-6), 166.20 (C-1), 94.19 (C-8), 158.60 (C-9), 105.78 (C-10), 123.33 (C-1'), 116.23 (C-2'), 145.97 (C-3'), 150.10 (C-4'), 117.92 (C-5'), 123.07 (C-6'), 105.52 (C-1"), 73.32 (C-2"), 75.24 (C-3"), 70.18 (C-4"), 77.34 (C-5"), 62.09 (C-6")이었다. 한편, 이들 측정값은 논문 [Lu, Y.R. and Foo, L.Y. Identification and quantification of major polyphenols in apple pomace., Food Chemistry, 59(2), 187-194 (1997)] 에 보고된 퀘서틴 3-O-β-D-갈락토피라노시드의 측정값과 일치하였다.Active substance 2 measured 1 H-NMR (500 MHz, CD 3 OD) analyzed by unit INOVA 500, δ6.16 (1H, d, J = 2.0 Hz, H-6), 6.37 ( 1H, d, J = 2.0 Hz, H-8), 7.84 (1H, d, J = 2.0 Hz, H-2 '), 6.86 (1H, d, J = 8.5 Hz, H-5'), 7.58 ( 1H, dd, J = 2.0, 8.5 Hz, H-6 '), 5.12 (1H, d, J = 8.0 Hz, H-1 "), 4.55-3.33 (H-2" to H-6 "), Measurements analyzed by 13 C-NMR (125 MHz, CD 3 OD) are δ 158.94 (C-2), 135.91 (C-3), 179.90 (C-4), 163.18 (C-5), 100.20 ( C-6), 166.20 (C-1), 94.19 (C-8), 158.60 (C-9), 105.78 (C-10), 123.33 (C-1 '), 116.23 (C-2'), 145.97 (C-3 '), 150.10 (C-4'), 117.92 (C-5 '), 123.07 (C-6'), 105.52 (C-1 "), 73.32 (C-2"), 75.24 (C -3 "), 70.18 (C-4"), 77.34 (C-5 "), 62.09 (C-6"). On the other hand, these measurements were reported in the literature [Lu, YR and Foo, LY Identification and quantification of major polyphenols in apple pomace., Food Chemistry, 59 (2), 187-194 (1997), consistent with the measurements of quercetin 3- O- β-D-galactopyranoside reported.

이상의 MS 및 NMR등의 기기분석결과로부터 이 물질의 구조를 하기와 같이 결정하였다.From the results of instrumental analysis such as MS and NMR, the structure of this material was determined as follows.

4) 활성물질 3(퀘서틴 3-O-β-D-글루코피라노시드)의 구조해석4) Structural Analysis of Active Substance 3 (Quercetin 3- O- β-D-Glucopyranoside)

활성물질 3은 기기(UnitINOVA 500)에 의해 분석된 1H-NMR (500 MHz, CD3 OD)의 측정값은 δ6.16 (1H, d, J = 2.0 Hz, H-6), 6.37 (1H, d, J = 2.0 Hz, H-8), 7.84 (1H, d, J = 2.0 Hz, H-2'), 6.86 (1H, d, J = 8.5 Hz, H-5'), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 5.26 (1H, d, J = 7.5 Hz, H-1"), 4.55-3.33 (H-2"∼H-6")이며, 13C-NMR (125 MHz, CD3OD)의 측정값은 δ 158.94 (C-2), 135.91 (C-3), 179.90 (C-4), 163.18 (C-5), 100.02 (C-6), 166.20 (C-7), 94.84 (C-8), 158.60 (C-9), 105.78 (C-10), 123.33 (C-1'), 116.14 (C-2'), 145.97 (C-3'), 150.10 (C-4'), 117.69 (C-5'), 123.02 (C-6'), 104.42 (C-1"), 75.88 (C-2"), 78.56 (C-3"), 71.37 (C-4"), 78.56 (C-5"), 62.69 (C-6")이었다. 활성물질 3의 1H- 및 13C-NMR의 측정값은 Lu 등의 논문에서 보고된 퀘서틴 3-O-β-D-글루코피라노시드의 측정값과 일치하였다. 또한 Lu 등에 의하면 활성물질 2와 3은 HPLC 상에서 동일용출을 갖는 것으로 알려졌다.Active substance 3 measured 1 H-NMR (500 MHz, CD 3 OD) analyzed by unit INOVA 500, δ6.16 (1H, d, J = 2.0 Hz, H-6), 6.37 ( 1H, d, J = 2.0 Hz, H-8), 7.84 (1H, d, J = 2.0 Hz, H-2 '), 6.86 (1H, d, J = 8.5 Hz, H-5'), 7.58 ( 1H, dd, J = 2.0, 8.5 Hz, H-6 '), 5.26 (1H, d, J = 7.5 Hz, H-1 "), 4.55-3.33 (H-2" to H-6 "), 13 C-NMR (125 MHz, CD 3 OD) measured δ 158.94 (C-2), 135.91 (C-3), 179.90 (C-4), 163.18 (C-5), 100.02 (C-6 ), 166.20 (C-7), 94.84 (C-8), 158.60 (C-9), 105.78 (C-10), 123.33 (C-1 '), 116.14 (C-2'), 145.97 (C- 3 '), 150.10 (C-4'), 117.69 (C-5 '), 123.02 (C-6'), 104.42 (C-1 "), 75.88 (C-2"), 78.56 (C-3 " ), 71.37 (C-4 "), 78.56 (C-5"), 62.69 (C-6 "). Measurements of 1 H- and 13 C-NMR of active substance 3 were reported in Lu et al. The results were consistent with the measurements of quercetin 3- O- β-D-glucopyranoside, and according to Lu et al., Active substances 2 and 3 were found to have the same elution on HPLC.

이상의 MS 및 NMR 등의 기기분석결과로부터 이 물질의 구조를 하기와 같이 결정하였다.From the results of instrumental analysis such as MS and NMR, the structure of this material was determined as follows.

5) 활성물질 4(퀘서틴 3-O-α-D-아라비노피라노시드)의 구조해석5) Structural Analysis of Active Substance 4 (Quercetin 3- O- α-D-Arabinopyranoside)

활성물질 4를 구조해석하기 위하여 질량분석기(FAB-MS positive, Platform 2)로 분석을 행한 결과, [M+H]+으로 m/z 435가 검출되어 분자량이 434임을 알 수 있었다. 기기(UnitINOVA 500)에 의해 분석된 1H-NMR (500 MHz, CD3OD)의 측정값은 δ 6.20 (1H, d, J = 2.0 Hz, H-6), 6.40 (1H, d, J = 2.0 Hz, H-8), 7.75 (1H, d, J = 2.0 Hz, H-2'), 6.87 (1H, d, J = 8.5 Hz, H-5'), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 5.16 (1H, d, J = 6.5 Hz, H-1"), 4.55-3.33 (H-2"∼H-6")이며, 또 13 C-NMR (125 MHz, CD3OD)의 측정값은 δ158.9 (C-2), 135.8 (C-3), 179.6 (C-4), 163.2 (C-5), 100.0 (C-6), 166.1 (C-7), 94.8 (C-8), 158.6 (C-9), 105.8 (C-10), 123.0 (C-1'), 117.6 (C-2'), 146.1 (C-3'), 150.1 (C-4'), 116.3 (C-5'), 123.2 (C-6'), 104.8 (C-1"), 73.0 (C-2"), 74.3 (C-3"), 69.3 (C-4"), 67.1 (C-5")이었다. 그리고 활성물질 4의 13C-NMR 측정값은 논문 [De Almeida, A. P. et al., flavonol monoglycosides isolated from the antiviral fractions of Persea americana (Lauraceae) leaf infusion. Phytother. Res., 12, 8, 562-567, 1998] 에 보고된 퀘서틴 3-O-α-D-아라비노피라노시드의 13C-NMR 측정값과 일치하였다.Mass spectrometry (FAB-MS positive, Platform 2) to analyze the active material 4 as a result, it was found that the molecular weight is 434 by m / z 435 is detected with [M + H] + . The measured value of 1 H-NMR (500 MHz, CD 3 OD) analyzed by the instrument ( Unit INOVA 500) is δ 6.20 (1H, d, J = 2.0 Hz, H-6), 6.40 (1H, d, J = 2.0 Hz, H-8), 7.75 (1H, d, J = 2.0 Hz, H-2 '), 6.87 (1H, d, J = 8.5 Hz, H-5'), 7.58 (1H, dd, J = 2.0, 8.5 Hz, H-6 '), 5.16 (1H, d, J = 6.5 Hz, H-1 "), 4.55-3.33 (H-2" to H-6 "), and 13 C-NMR Measurements at (125 MHz, CD 3 OD) are δ 158.9 (C-2), 135.8 (C-3), 179.6 (C-4), 163.2 (C-5), 100.0 (C-6), 166.1 (C-7), 94.8 (C-8), 158.6 (C-9), 105.8 (C-10), 123.0 (C-1 '), 117.6 (C-2'), 146.1 (C-3 ') , 150.1 (C-4 '), 116.3 (C-5'), 123.2 (C-6 '), 104.8 (C-1 "), 73.0 (C-2"), 74.3 (C-3 "), 69.3 (C-4 "), 67.1 (C-5"). And 13 C-NMR measurements of activator 4 are described in De Almeida, AP et al ., Flavonol monoglycosides isolated from the antiviral fractions of Persea americana (Lauraceae) leaf infusion. Phytother. Res., 12, 8, 562-567, 1998, which is consistent with 13 C-NMR measurements of quercetin 3- O- α-D-arabinopyranoside.

이상의 MS 및 NMR등의 기기분석결과로부터 활성물질의 구조를 하기와 같이 결정하였다.From the results of instrumental analysis such as MS and NMR, the structure of the active material was determined as follows.

6) 활성물질 5(캠펠로 3-O-α-D-아라비노피라노시드)의 구조해석6) Structural Analysis of Active Substance 5 (Campello 3- O- α-D-Arabinopyranoside)

활성물질 5를 구조해석하기 위하여 질량분석기(FAB-MS positive, Platform 2)로 분석을 행한 결과, [H+Na]+로 m/z 441이 관측되어 이 활성물질의 분자량은 418임을 알 수 있었다. 기기(UnitINOVA 500)에 의해 분석된 1H-NMR (500 MHz, CD3 OD)의 측정값은 δ6.21 (1H, d, J = 2.0 Hz, H-6), 6.41 (1H, d, J = 2.0 Hz, H-8), 8.07 (1H, d, J = 9.0 Hz, H-2'), 7.89 (1H, d, J = 9.0 Hz, H-3'), 7.89 (1H, d, J = 9.0 Hz, H-5'), 8.07 (1H, d, J = 9.0 Hz, H-6'), 5.15 (1H, d, J = 6.5 Hz, H-1"), 4.55-3.33 (H-2"∼H-6")이며, 13C-NMR (125 MHz, CD3OD)의 측정값은 δ 158.63 (C-2), 135.80 (C-3), 178.30 (C-4), 163.26 (C-5), 100.02 (C-6), 166.16 (C-7), 94.87 (C-8), 158.90 (C-9), 105.70 (C-10), 123.10 (C-1'), 132.43 (C-2'), 116.40 (C-3'), 159.90 (C-4'), 116.40 (C-5'), 132.43 (C-6'), 104.46 (C-1"), 72.90 (C-2"), 74.13 (C-3"), 69.07 (C-4"), 66.87 (C-5")이었다. 또한 HMBC 분석에서 당 1위(H-1")와 켐펠로의 탄소 3위(C-3)에 크로스피크가 관찰되어 당의 결합위치를 알 수 있었다.Mass spectrometry (FAB-MS positive, Platform 2) was used to analyze the active substance 5 , and m / z 441 was observed with [H + Na] + , indicating that the active substance had a molecular weight of 418. . Measurements of 1 H-NMR (500 MHz, CD 3 OD) analyzed by the instrument ( Unit INOVA 500) were δ6.21 (1H, d, J = 2.0 Hz, H-6), 6.41 (1H, d, J = 2.0 Hz, H-8), 8.07 (1H, d, J = 9.0 Hz, H-2 '), 7.89 (1H, d, J = 9.0 Hz, H-3'), 7.89 (1H, d, J = 9.0 Hz, H-5 '), 8.07 (1H, d, J = 9.0 Hz, H-6'), 5.15 (1H, d, J = 6.5 Hz, H-1 "), 4.55-3.33 (H -2 "to H-6"), and measured values of 13 C-NMR (125 MHz, CD 3 OD) are 158.63 (C-2), 135.80 (C-3), 178.30 (C-4), and 163.26. (C-5), 100.02 (C-6), 166.16 (C-7), 94.87 (C-8), 158.90 (C-9), 105.70 (C-10), 123.10 (C-1 '), 132.43 (C-2 '), 116.40 (C-3'), 159.90 (C-4 '), 116.40 (C-5'), 132.43 (C-6 '), 104.46 (C-1 "), 72.90 (C -2 "), 74.13 (C-3"), 69.07 (C-4 "), 66.87 (C-5"). In the HMBC analysis, the peaks of the sugars (H-1 ") and Cempello's carbon third (C-3) were observed, and the peaks of the sugars were found.

이상의 MS, NMR등의 기기분석결과로부터 이 물질의 구조를 하기와 같이 결정하였다.From the results of instrumental analysis of MS and NMR, the structure of this material was determined as follows.

7) 활성물질 6(루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드)의 구조해석7) Structural Analysis of Active Substance 6 (Luteolin 3- O- β-D-Ranmopyranosyl (1 → 6) -7- O- α-L-Glucopyranoside)

활성물질 6을 구조해석하기 위하여 질량분석기(FAB-MS positive, JMS-HX/HX110A Tandem Mass Spectrometer, Jeol, Japan)로 분석을 행한 결과, 분자이온 피크로 [M+H]+으로 m/z 595가 검출되어 분자량이 594임을 알 수 있었다. 기기(UnitINOVA 500)에 의해 분석된 1H-NMR (500 MHz, pyridine-d 5 )의 측정값은 δ 6.87 (1H, s, H-3), 6.82 (1H, d, J = 2.0 Hz, H-6), 6.97 (1H, d, J = 2.0 Hz, H-8), 7.99 (1H, d, J = 2.0Hz, H-2'), 7.38 (1H, d, J = 8.5Hz, H-5'), 7.56 (1H, dd, J = 2.0, 8.5Hz, H-6'), 5.69 (1H, d, J = 7.5Hz, H-1"), 4.32 (1H, dd, J = 7.5, 9.0 Hz, H-2"), 4.30 (1H, dd, J = 3.5, 9.0 Hz, H-3"), 4.66 (1H, dd, J = 3.5, 9.0 Hz, H-4"), 4.40 (1H, dd, J = 9.0, 9.0 Hz, H-5"), 4.16 (1H, d, J = 9.0 Hz, H-6"a), 4.76 (1H, d, J = 7.0 Hz, H-6"b), 5.51 (1H, s, H-1"'), 4.75 (1H, s, H-2"'), 4.13 (1H, dd, J = 9.5, 9.5 Hz, H-3"'), 4.27 (1H, dd, J = 9.5, 9.5 Hz, H-4"'), 4.34 (1H, dd, J = 6.0, 9.5 Hz, H-5"'), 1.61 (1H, d, J = 6.0 Hz, H-6"')이며, 13C-NMR (125 MHz, pyridine-d 5 )의 측정값은 δ166.04 (C-2), 104.78 (C-3), 183.26 (C-4), 163.06 (C-5), 101.33 (C-6), 164.60 (C-7), 95.65 (C-8), 158.46 (C-9), 107.21 (C-10), 123.44 (C-1'), 115.19 (C-2'), 148.10 (C-3'), 152.35 (C-4'), 117.60 (C-5'), 120.45 (C-6'), 102.69 (C-1"), 75.24 (C-2"), 78.07 (C-3"), 72.57 (C-4"), 78.87 (C-5"), 68.17 (C-6"), 102.69 (C-1"'), 73.40 (C-2"'), 71.95 (C-3"'), 74.49 (C-4"'), 70.39 (C-5"'), 19.06 (C-6"')이었다. 또한, HMBC 분석에서 글루코스 1위(H-1")와 luteolin 7위(C-7), 그리고 람노스 1위(H-1"')와 글루코스 6위(C-6")간에 크로스피크가 관찰되어 당들의 결합위치를 알 수 있었다.To analyze the active substance 6 , a mass spectrometer (FAB-MS positive, JMS-HX / HX110A Tandem Mass Spectrometer, Jeol, Japan) analyzed the molecular ion peak [M + H] + to m / z 595. Was detected and it was found that the molecular weight was 594. Measurements of 1 H-NMR (500 MHz, pyridine- d 5 ) analyzed by the instrument ( Unit INOVA 500) were δ 6.87 (1H, s, H-3), 6.82 (1H, d, J = 2.0 Hz, H-6), 6.97 (1H, d, J = 2.0 Hz, H-8), 7.99 (1H, d, J = 2.0 Hz, H-2 '), 7.38 (1H, d, J = 8.5 Hz, H -5 '), 7.56 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 5.69 (1H, d, J = 7.5 Hz, H-1 "), 4.32 (1H, dd, J = 7.5 , 9.0 Hz, H-2 "), 4.30 (1H, dd, J = 3.5, 9.0 Hz, H-3"), 4.66 (1H, dd, J = 3.5, 9.0 Hz, H-4 "), 4.40 ( 1H, dd, J = 9.0, 9.0 Hz, H-5 "), 4.16 (1H, d, J = 9.0 Hz, H-6" a), 4.76 (1H, d, J = 7.0 Hz, H-6 " b), 5.51 (1H, s, H-1 "'), 4.75 (1H, s, H-2"'), 4.13 (1H, dd, J = 9.5, 9.5 Hz, H-3 "'), 4.27 (1H, dd, J = 9.5, 9.5 Hz, H-4 "'), 4.34 (1H, dd, J = 6.0, 9.5 Hz, H-5"'), 1.61 (1H, d, J = 6.0 Hz, H-6 "'), and the measurements of 13 C-NMR (125 MHz, pyridine- d 5 ) are δ166.04 (C-2), 104.78 (C-3), 183.26 (C-4), 163.06 ( C-5), 101.33 (C-6), 164.60 (C-7), 95.65 (C-8), 158.46 (C-9), 107.21 (C-10), 123.44 (C-1 '), 115.19 ( C-2 '), 148.10 (C-3'), 152.35 (C-4 '), 117.60 (C-5'), 120.45 (C-6 '), 102.69 (C-1 "), 75.24 (C-2"), 78.07 (C-3 "), 72.57 (C-4"), 78.87 (C-5 "), 68.17 (C-6"), 102.69 (C -1 "'), 73.40 (C-2"'), 71.95 (C-3 "'), 74.49 (C-4"'), 70.39 (C-5 "'), 19.06 (C-6"') It was. In addition, cross-peak peaks were found between the 1st glucose (H-1 ") and the 7th luteolin (C-7), and the 1st (R-1"') and 6th glucose (C-6 ") of Rhamnose in HMBC analysis. Observation revealed the location of the sugar binding.

이상의 MS, NMR등의 기기분석결과로부터 이 물질의 구조를 하기와 같이 결정하였다.From the results of instrumental analysis of MS and NMR, the structure of this material was determined as follows.

실시예 3 : 신선초조제물에 함유된 물질들의 항산화 활성Example 3 Antioxidant Activity of Substances in Fresh Herbal Preparations

분리된 활성물질들의 항산화 활성은 문 등의 방법 [Moon J.H. et al., Antioxidant activity of caffeic acid and digydrocaffeic Acid in lard and human low-density lipoprotein, J. Agric, Food Chem., 46, 2, 5062-5065, 1998] 에 의해 활성물질의 1분자당 DPPH 라디칼소거수를 측정하였다. 10 mM의 트리스-염산완충용액 [pH 7.4, tris-HCl buffer solution (Sigma, USA)] 1 mL를 적당한 수의 갈색 시험관에 취한 후, 메탄올에 용해한 항산화제를 최종농도가 1 μM에서부터 100 μM이 되도록 시험관에 가하였다. 그리고 각 용액이 1.5 mL가 되도록 메탄올을 가한 후, 400 μM의 DPPH 에탄올 용액 (최종농도, 100 μM) 0.5 mL를 각 용액에 2분 간격으로 순차 첨가하고, 가볍게 혼합한 다음 실온에서 30분간 반응시켜, DPPH 첨가순에 따라 2분 간격으로 517 nm에서 흡광도를 측정하여 흡광도가 더 이상 변화하지 않는 최초의 농도를 측정하여 단리된 화합물 1분자당의 DPPH 라디칼소거수를 결정하였다. 또 대표적인 지용성 항산화제인 알파토코페롤과 분리된 화합물들의 아글리콘인 루테올린 (Sigma, USA), 퀘서틴 (Sigma, USA), 캠펠로 (Sigma, USA)등을 비교구로 하여 측정하였다. 이들 물질에 대한 항산화 활성결과는 알파토코페를 1분자가 DPPH 라디칼 2.0분자를 소거한다는 것을 전제로 하여 계산하였다. 그 결과, DPPH 라디칼소거에 대한 항산화 활성은 퀘서틴 (6.7분자) > 퀘서틴 3-O-β-D-갈락토피라노시드 (4.2분자) 〓 퀘서틴 3-O-β-D-글루코피라노시드 (4.2분자) ≥퀘서틴 3-O-α-D-아라비노피라노시드 (4.1분자) > 루테올린 (2.9분자) > 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드 (2.5분자) 〓 캠펠로 (2.5분자) > 루테올린 7-O-β-D-글루코퍼라노시드 ( 2.2분자) > 캠펠로 3-O-α-D-아라비노피라노시드 (활성물질 5, 1.4분자) 순으로 나타냈다. 신선초지상부의 건조분말에서 분리한 활성물질들은 강한 항산화 활성을 나타냈으며, 캔펠로 3-O-α-D-아라비노피라노시드를 제외한 모든 활성물질들은 대표적인 지용성 항산화제인 알파토코페롤보다 강함을 알 수 있었다.Antioxidant activity of the isolated actives was described by Moon JH et al ., Antioxidant activity of caffeic acid and digydrocaffeic Acid in lard and human low-density lipoprotein, J. Agric, Food Chem., 46, 2, 5062- 5065, 1998], the number of DPPH radical eliminations per molecule of active substance was measured. 1 mL of 10 mM tris-hydrochloric acid buffer solution [pH 7.4, tris-HCl buffer solution (Sigma, USA)] was taken in an appropriate number of brown test tubes, and antioxidants dissolved in methanol were added at a final concentration of 1 μM to 100 μM. It was added to the test tube as much as possible. Methanol was added so that each solution was 1.5 mL, and 0.5 mL of 400 μM DPPH ethanol solution (final concentration, 100 μM) was sequentially added to each solution at 2 minute intervals, mixed gently, and reacted at room temperature for 30 minutes. , DPPH radical elimination number per molecule of the isolated compound was determined by measuring the absorbance at 517 nm at intervals of 2 minutes according to the DPPH addition order. In addition, typical lipid-soluble antioxidants, alphatocopherol and aglycone, luteolin (Sigma, USA), quercetin (Sigma, USA), and campello (Sigma, USA), which were isolated compounds, were measured by comparison. The antioxidant activity results for these substances were calculated on the assumption that one molecule of alphatocope eliminated 2.0 molecules of DPPH radicals. As a result, the antioxidant activity against DPPH radical scavenging was found to be: Quercetin (6.7 molecules)> Quesertin 3- O- β-D-galactopyranoside (4.2 molecules) 〓 Quesertin 3- O- β-D-glucopy Lanoside (4.2 molecule) ≥Quercetin 3- O- α-D-arabinopyranoside (4.1 molecule)> Luteolin (2.9 molecule)> Luteolin 3- O- β-D-rhamnopyranosyl (1 → 6) -7- O- α-L-glucopyranoside (2.5 molecules) 〓 Campello (2.5 molecules)> Luteolin 7- O- β-D-glucofuranoside (2.2 molecules)> Campello 3- O -α-D-arabinofyranoside (active substance 5 , 1.4 molecules) was shown in order. The active materials isolated from the dry powder of fresh grass showed strong antioxidant activity, and all the active materials except for Canpelo 3- O- α-D-arabinopyranoside were stronger than the representative fat-soluble antioxidant alphatocopherol. there was.

본 발명에서 천연항산화물질을 포함한 신선초조제물에는 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드 등의 항산화활성을 갖는 물질이 존재함을 발견하였으며, 이들 물질은 항산화 및 노화억제 효력을 갖는 라디칼 소거기능을 갖고 있음이 검정되었다. 이들 천연 항산화 활성물질이 포함된 신선초조제물은 산화에 의한 질병예방 효과 및 노화억제 효과를 갖는 기능성 소재로 이용이 기대된다.Fresh preparations containing natural antioxidants in the present invention include luteolin 7- O- β-D-glucopyranoside, quercetin 3- O- β-D-galactopyranoside, quercetin 3- O- β -D- glucopyranoside, Quebec seotin 3- O -α-D- arabino pyrano seed, fellow cam 3- O -α-D- arabino pyrano seed, luteolin 3- O -β-D- It was found that a substance having antioxidant activity such as rhamnopyranosyl (1 → 6) -7- O- α-L-glucopyranoside was present, and these substances have a radical scavenging function having antioxidant and anti-aging effects. Yes was tested. Fresh herbal preparations containing these natural antioxidant actives are expected to be used as functional materials having the effect of preventing disease by oxidization and anti-aging effect.

Claims (3)

항산화 기능을 갖는 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드 등의 천연항산화물질을 포함하여 항산화 기능을 갖는 신선초조제물.Luteolin 7- O- β-D-glucopyranoside with antioxidant function, quercetin 3- O- β-D-galactopyranoside, quercetin 3- O- β-D-glucopyranoside, Quercetin 3- O- α-D-arabinopyranoside, Campello 3- O- α-D-arabinopyranoside, luteolin 3- O- β-D-ramnopyranosyl (1 → 6) A fresh herb preparation having an antioxidant function, including natural antioxidants such as -7- O- α-L-glucopyranoside. 제 1항에 있어서 신선초조제물은, 신선초지상부를 동결건조방법으로 분말화하여 제조된 것임을 특징으로 하는 신선초조제물.The fresh herb preparation according to claim 1, wherein the fresh herb preparation is prepared by pulverizing the fresh grass top part by a lyophilization method. 제 1항에 있어서 항산화물질은, 신선초지상부의 건조분말을 열수로 추출하여 얻는 단계, 용매분획하여 에틸아세테이트가용중성분획물을 얻는 단계, 에틸아세테이트가용중성획분을 흡착을 이용한 세파덱스 LH-20 컬럼 크로마토그라피로 분획하는 단계, 또한 역상컬럼이 장착된 HPLC로 메탄올-물 용매계에 의하여 에틸아세테이트가용중성획분으로부터 루테올린 7-O-β-D-글루코피라노시드, 퀘서틴 3-O-β-D-갈락토피라노시드, 퀘서틴 3-O-β-D-글루코피라노시드, 퀘서틴 3-O-α-D-아라비노피라노시드, 캠펠로 3-O-α-D-아라비노피라노시드, 루테올린 3-O-β-D-람노피라노실(1→6)-7-O-α-L-글루코피라노시드를 분리하는 단계를 포함한 방법에 의해 이들 천연 항산화물질이 분리되고 기기분석등에 의해 동정된 이들 물질이 포함되어 있음이 확인된 신선초조제물.The method of claim 1, wherein the antioxidant is obtained by extracting the dry powder of fresh grassland with hot water, fractionation of solvent to obtain ethyl acetate soluble component fraction, Sepadex LH-20 column chromatography using ethyl acetate soluble neutral fraction by adsorption. Fractionation by graphiography, and also luteolin 7- O- β-D-glucopyranoside, quercetin 3- O -β- from ethylacetate soluble neutral fraction by methanol-water solvent system by HPLC equipped with reverse phase column. D-galactopyranoside, quercetin 3- O- β-D-glucopyranoside, quercetin 3- O- α-D-arabinofyranoside, campello 3- O- α-D-ara These natural antioxidants were separated by a method comprising the step of separating the vinopyranoside, luteolin 3- O- β-D-ramnopyranosyl (1 → 6) -7- O- α-L-glucopyranoside. Fresh preparations that have been identified to contain these substances that have been isolated and identified by instrumental analysis.
KR1020040011177A 2004-02-16 2004-02-16 Angelica keiskei preparation for antioxidative function containing Inteolin 7-O-β-D-glucopyranoside, quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-galactopyranoside, quercetin 3-O-α-D-arabinopyranoside, kaempferol 3-O-α-D-arabinopyranoside, luteolin 3-O-β-D-rhamnopyranosyl(1→6)-7-O-α-L-glucopyranoside and preparation method thereof KR20050081810A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102304160A (en) * 2011-01-29 2012-01-04 沈阳双鼎制药有限公司 Preparation method and application of luteolin-7-O-gentibioside
CN104198636A (en) * 2014-08-15 2014-12-10 中国农业科学院蜜蜂研究所 Method for identifying bee pollen on basis of content of isoquercitrin
KR20160080623A (en) * 2014-12-30 2016-07-08 재단법인 경기과학기술진흥원 Cosmetic composition comprising luteolin-6-c-fucopyranoside having skin whitening and anti-aging activity
CN108997457A (en) * 2018-07-10 2018-12-14 宁夏医科大学 Galuteolin and Quercetin -3-o- β-glucopyranoside extracting method in Chinese white poplar leaf
CN111638299A (en) * 2020-07-06 2020-09-08 广西中医药大学 Method for establishing Angelica keiskei high-performance liquid chromatography fingerprint

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102304160A (en) * 2011-01-29 2012-01-04 沈阳双鼎制药有限公司 Preparation method and application of luteolin-7-O-gentibioside
CN104198636A (en) * 2014-08-15 2014-12-10 中国农业科学院蜜蜂研究所 Method for identifying bee pollen on basis of content of isoquercitrin
CN104198636B (en) * 2014-08-15 2015-11-18 中国农业科学院蜜蜂研究所 A kind of method differentiating Bee Pollen based on Isoquercitrin content
KR20160080623A (en) * 2014-12-30 2016-07-08 재단법인 경기과학기술진흥원 Cosmetic composition comprising luteolin-6-c-fucopyranoside having skin whitening and anti-aging activity
CN108997457A (en) * 2018-07-10 2018-12-14 宁夏医科大学 Galuteolin and Quercetin -3-o- β-glucopyranoside extracting method in Chinese white poplar leaf
CN111638299A (en) * 2020-07-06 2020-09-08 广西中医药大学 Method for establishing Angelica keiskei high-performance liquid chromatography fingerprint

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