KR20170025977A - Novel Chemical Isolated From Oxyrrhis marina - Google Patents

Novel Chemical Isolated From Oxyrrhis marina Download PDF

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KR20170025977A
KR20170025977A KR1020150123156A KR20150123156A KR20170025977A KR 20170025977 A KR20170025977 A KR 20170025977A KR 1020150123156 A KR1020150123156 A KR 1020150123156A KR 20150123156 A KR20150123156 A KR 20150123156A KR 20170025977 A KR20170025977 A KR 20170025977A
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compound
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compounds
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박재연
윤은영
정해진
노정래
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재단법인차세대융합기술연구원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/03—Monocarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/08—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium
    • C07H5/10—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium to sulfur

Abstract

The present invention relates to novel compounds isolated from Oxyrrhis marina and, more specifically, to a novel oxylipin-based compound represented by chemical formula 1 and a novel DHA derivative compound represented by chemical formula 2. The novel isolated two compounds of the present invention have no toxicity and may have various activities compared to an existing similar compound.

Description

옥시리스 마리나로부터 분리된 신규 화합물{Novel Chemical Isolated From Oxyrrhis marina}Novel Chemical Isolated from Oxyrrhis marina < RTI ID = 0.0 >

본 발명은 와편모조류 Oxyrrhis marina로부터 분리된 신규 DHA 유도체 화합물에 대한 것이다.The present invention is directed to novel DHA derivative compounds isolated from the epidemic bird Oxyrrhis marina .

옥시리스속(Oxyrrhis)은 와편모충류 속의 하나이다. 옥시리스 마리나(Oxyrrhis marina) 등을 포함하고 있다.Oxyrrhis (Oxyrrhis) is a member of the genus Corynebacterium. And the Oxyrrhis marina.

와편모충류(渦鞭毛蟲類, dinoflagellates)는 대형 편모충류 원생생물 분류군의 하나이다. 대부분 해양 플랑크톤이지만, 담수 환경에서도 발견된다. 분포하는 개체수는 해수면 온도와 염도 또는 수심 등에 의존한다. 모든 와편모충류의 약 절반은 광합성을 하며, 이들은 규조류를 제외하고 해양 진핵생물 조류 중에서 가장 큰 분류군을 형성한다.And dinoflagellates are one of the larvae of the protoplasm family. Most are marine plankton, but are also found in freshwater environments. The number of populations distributed depends on sea level temperature, salinity or depth. Approximately half of all species and species are photosynthetic, and they form the largest taxa of marine eukaryotic algae except diatoms.

현재, 약 1,555종의 자유 생활하는 와편모충류를 기술하고 있다. 또한 1,700종 이상의 해양 종과 220여 종의 담수 종을 포함하여 약 2,000여 종으로 추산하기도 한다. 가장 크게 추산하는 경우는 현존하 와편모충류를 2,294종으로 집계하며, 해수와 담수 그리고 기생하는 와편모충류를 포함하고 있다.Currently, there are about 1,555 species living freely and describing species. It is estimated to be about 2,000 species, including more than 1,700 marine species and 220 freshwater species. The highest estimates include 2,294 species in the subfamily, including seawater, freshwater, and parasitic species.

최근 와편모충류로부터 유용한 화합물을 분리하고자 하는 시도가 확인되고 있다.Attempts have recently been made to isolate useful compounds from P. albicans.

본 발명은 옥시리스 마리나(Oxyrrhis marina)로부터 분리한 유용한 화합물을 제공하는데 목적이 있다.It is an object of the present invention to provide useful compounds isolated from Oxyrrhis marina.

본 발명은 하기 화학식 1로 나타나는 신규의 oxylipin계열의 화합물을 제공한다.The present invention provides novel oxylipin compounds represented by the following general formula (1).

[화학식 1][Chemical Formula 1]

Figure pat00001
Figure pat00001

본 발명은 하기 화학식 1로 나타나는 신규의 DHA 유도체 화합물을 제공한다.The present invention provides a novel DHA derivative compound represented by the following formula (1).

[화학식 2](2)

Figure pat00002
Figure pat00002

본 발명의 분리된 신규한 화합물은 분리된 두 화합물은 독성이 없으며 기존 유사 화합물로부터 판단할 때 다양한 활성을 가질 것으로 예상된다.The isolated novel compounds of the present invention are expected to have a variety of activities as judged from existing analogous compounds without the two compounds being toxic.

본 발명의 도면은 신규한 화합물을 확인하기 위한 실험결과이다.The drawings of the present invention are experimental results for identifying novel compounds.

본 발명은 본 발명은 하기 화학식 1로 나타나는 신규의 oxylipin계열의 화합물을 제공한다.The present invention provides novel oxylipin-based compounds represented by the following general formula (1).

[화학식 1][Chemical Formula 1]

Figure pat00003
Figure pat00003

본 발명은 하기 화학식 1로 나타나는 신규의 DHA 유도체 화합물을 제공한다.The present invention provides a novel DHA derivative compound represented by the following formula (1).

[화학식 2](2)

Figure pat00004
Figure pat00004

본 발명의 분리된 신규한 화합물은 분리된 두 화합물은 독성이 없으며 기존 유사 화합물로부터 판단할 때 다양한 활성을 가질 것으로 예상된다.The isolated novel compounds of the present invention are expected to have a variety of activities as judged from existing analogous compounds without the two compounds being toxic.

실험방법Experimental Method

실험방법 Experimental Method 1.화합물1. Compound 분리 detach

와편모조류 Oxyrrhis marina를 1000 L규모로 실험실에서 배양한 후 원심분리기를 사용하여 세포를 수확하였다. 수확된 세포에 메탄올 용매를 사용하여 2일간 추출하여 약 10 g의 조추출물을 얻었다. 조추출물은 염을 제거하기 위하여 물과 염화메틸렌으로 분액깔때기를 사용하여 분배한 후 염화메틸렌 층 2g을 확보하였다. 다시 이 분액을 헥산과 혼합 극성용매 (85% 메탄올과 15% 물)로 재분배하여 극성 분액 900 mg을 얻었다. 이 분액은 SEC(size exclusion chromatography, column 100 Å + 50 Å) 를 거쳐 9개의 분액으로 나누었으며 그 중 25분에서 얻어진 분액 (M6)과 35분에서 얻어진 분액에서 (M7~ M9) glycolipid함량이 높았다. 먼저 M6에 대하여 glycolipid를 분리하기 위하여 HPLC를 이용하였다. HPLC 분리 조건은 Synergi사의 polar-RP 컬럼 (250 mm × 4.6 mm, 5 νm)를 사용하였으며 전개용매는 메탄올과 물을 사용하여 35분 동안 초기 60% 메탄올에서 100% 메탄올까지 증가시켰다. 유속은 0.8 ml/min하여 18분경 정제된 화합물 1 (10 mg)을 얻었다. 그리고 화합물 2는 세 분액 (M7, M8, M9)를 합친 후 동일한 방법으로 HPLC를 수행하여 26분경에 약 13mg을 얻었다. Oxyrrhis The marina was cultivated in a laboratory at 1000 L, and cells were harvested using a centrifuge. The harvested cells were extracted with methanol solvent for 2 days to obtain about 10 g crude extract. The crude extract was partitioned with water and methylene chloride using a separatory funnel to remove salts and 2 g of methylene chloride layer was obtained. Again, this fraction was redistributed with hexane and a mixed polar solvent (85% methanol and 15% water) to obtain a polar fraction (900 mg). This fraction was divided into nine fractions by SEC (size exclusion chromatography, column 100 Å + 50 Å), and glycolipid content was high in the fraction (M6) obtained at 25 minutes and in the fraction obtained at 35 minutes (M7 to M9) . First, HPLC was used to separate glycolipids from M6. HPLC separation was performed using Synergi's polar-RP column (250 mm × 4.6 mm, 5 νm) and the developing solvent was increased from 60% methanol to 100% methanol for 35 minutes using methanol and water. The flow rate was 0.8 ml / min, and purified Compound 1 (10 mg) was obtained in about 18 minutes. Compound 2 was obtained by collecting three fractions (M7, M8, and M9) and then performing HPLC by the same method to obtain about 13 mg at about 26 minutes.

실험방법 2. 세포 배양Experimental method 2. Cell culture

실험에 사용된 세포주 RAW264.7 대식세포는 한국세포주은행(KCLB, Korean Cell Line Bank, Seoul, Korea)에서 분양받아 사용하였다. RAW264.7 대식세포는 10% FBS, 100 units/mL penicillin과 50 ㎍/mL streptomycin을 함유한 DMEM 배지에서 2×106개의 세포를 직경 10 mm의 culture dish에 넣고 5% CO2가 공급되는 incubator에서 37oC로 배양하였다.The cell line RAW264.7 macrophage used in the experiment was purchased from the Korean Cell Line Bank (KCLB, Korean Cell Line Bank, Seoul, Korea). RAW264.7 macrophages 10% FBS, 100 units / mL penicillin and 50 ㎍ / mL into a 2 × 10 6 cells in DMEM medium containing streptomycin in the culture dish having a diameter of 10 mm 5% CO incubator 2 which is supplied And cultured at 37 ° C.

실험방법 3. 세포 독성 측정Experimental method 3. Cytotoxicity measurement

화합물 1의 세포 독성을 알아보기 위하여 MTT(3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyl tetrazolium bromide) assay를 이용하여 측정하였다. MTT(2 mg/10mL) 시약을 각 well에 20μL씩 첨가하고 2시간 동안 37°C incubator에서 반응시킨 후 MTT 시약이 함유된 배지를 제거하였다. 각 well에 200 μL의 DMSO를 첨가하여 MTT를 환원하여 형성된 fomazan을 녹여 ELISA reader를 이용하여 450 nm에서 흡광도를 측정하였다.To determine the cytotoxicity of Compound 1 , it was measured using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) assay. MTT (2 mg / 10 mL) reagent was added to each well in an amount of 20 μL, followed by incubation for 2 hours at 37 ° C in an incubator, and then the media containing the MTT reagent was removed. After adding 200 μL of DMSO to each well, the fomazan formed by reducing MTT was dissolved and absorbance was measured at 450 nm using an ELISA reader.

실험결과Experiment result

실험결과 Experiment result 1.화합물1. Compound 1 One 의of 구조 결정 Structure determination

화합물 1의 구조는 NMR 스펙트럼 해석으로 결정되었으며 질량 분석 데이터로부터 확인되었다. 화합물 1의 분자량은 ESI-LRMS에서 [M + Na]+ m/z = 401로부터 378로 주어졌다 [도 1]. 수소 NMR스펙트럼은 저자장 영역에서 이중결합에 해당되는 신호와 고자장에 일차 메틸기 한 개가 주어져 있으며 옥시 메틴기에 붙은 수소 신호 3개가 특징적으로 관찰되었다 [도 2]. 탄소 스펙트럼은 이중 결합에 해당되는 탄소 신호 10개, 산소와 결합된 탄소 3개 그리고 고자장 영역에 7개의 탄소가 관찰되었다 [도 3]. 하지만 탄소 스펙트럼에서는 매우 약한 신호로 주어져 있지만 HMBC 스펙트럼을 통하여 35.6 과 179.3 ppm에 두 개의 탄소가 더 존재한다는 것을 확인하였다. 따라서 화합물 1은 모두 22개의 탄소로 구성되어져 있었다. 먼저 COSY 스펙트럼을 분석한 결과 3.96 ppm 에 있는 메틴 수소 (H-10)는 3.54 ppm의 수소 (H-11)와 연결되어 있으며 또한 5.76 ppm에 있는 올레핀 수소 (H-9)와 연결되어 있었다. 다시 5.76 ppm에 있는 수소는 이와 매우 근접한 5.74 ppm의 수소 (H-8)와의 연결되어 있었는 데 이는 H-10과 136.2 ppm에 있는 탄소와의 HMBC 상관관계로부터 확인할 수 있었다. 계속하여 5.74 ppm에 있는 올레핀 수소는 남아 있는 옥시메틴기와 연결되어 있는 것을 COSY스펙트럼으로 확인할 수 있었다. COSY 스펙트럼은 계속하여 메틸렌-이중결합-메틸렌으로 이어져 있는 것을 보여주었다 [도 4]. 그리고 또한 2.32 ppm에 있는 메틸렌 (H-3)은 HMBC 실험에서 35.6 ppm에 있는 매우 약한 탄소(C-2)와의 상관관계 신호를 주었으며 이 탄소와 연결된 수소는 동일하게 2.32 ppm으로 관측되었다. 더 나아가 이들 두 수소는 다시 매우 약한 신호인 카보닐 탄소 (C-1)와의 HMBC 상관관계를 나타내었다. 한편 지금까지 설명되지 않은 신호는 6개의 이중결합 신호, 4개의 메틸렌 그리고 말단 메틸기가 있는 데 이들의 탄소 화학적 이동값으로 판단할 때 이중결합 사이 메틸렌이 한 개씩 배치된 선형 구조로 되어 있다는 추정할 수 있었으며 COSY 데이터로부터 확인되었다. 이들 선형 부분구조는 11번 탄소에 연결되어 있는 것을 3.54 ppm (H-11)과 2.18 ppm (H-12)의 수소와의 COSY 상관관계로부터 확인되었다. 그리고 8번 과 9번 탄소로 이루어진 이중결합은 이들 탄소에 붙은 수소들의 짝결합 상수값(J = 15.7 Hz)로부터 E형태로 결정되었고 나머지 4개의 이중결합은 탄소의 화학적 이동값으로 판단할 때 Z 형태로 주어졌다. 따라서 화합물 1의 구조는 (4Z, 8E, 13Z, 16Z, 19Z)-7, 10, 11-trihydroxydocosa-4,8,13,16,19-pentaenoic acid로 밝혀졌으며 화합물 검색 결과 이 구조는 현재까지 알려지지 않은 신규 구조이다. 이 구조를 MS/MS질량 분석한 결과 m/z 값들이 137, 149, 167, 205, 223로 측정되었는데 이들 분자 파편은 화합물 1의 구조로부터 해석될 수 있었다 [도 5]. 표 1은 화합물 1에 대한 수소와 탄소 NMR 데이터를 정리한 것이다.The structure of Compound 1 was determined by NMR spectrum analysis and confirmed from mass spectrometry data. The molecular weight of Compound 1 was given in [Epsilon] -LRMS as [M + Na] + m / z = 401 to 378 [Fig. In the hydrogen NMR spectrum, a signal corresponding to a double bond in the author region and a primary methyl group in a high magnetic field were given, and three hydrogen signals attached to the oxy methine group were characteristic (FIG. 2). The carbon spectrum showed 10 carbon signals corresponding to double bonds, 3 carbons bonded with oxygen and 7 carbons in the high magnetic field region (Fig. 3). However, it is confirmed that there are two carbon atoms in the HMBC spectrum at 35.6 and 179.3 ppm, although it is given as a very weak signal in the carbon spectrum. Thus, Compound 1 was composed of all 22 carbons. The analysis of the COZY spectrum revealed that the methine hydrogen (H-10) at 3.96 ppm was linked to 3.54 ppm hydrogen (H-11) and to the olefin hydrogen (H-9) at 5.76 ppm. Again, the hydrogen at 5.76 ppm was associated with 5.74 ppm hydrogen (H-8), which was very close to this, as confirmed by the HMBC correlation between H-10 and carbon at 136.2 ppm. It was confirmed by COZY spectrum that the olefin hydrogen at 5.74 ppm was connected to the remaining oxy methine group. The COZY spectrum was subsequently shown to be methylene-double bond-methylene (Fig. 4). Also, methylene (H-3) at 2.32 ppm gave a correlation signal with very weak carbon (C-2) at 35.6 ppm in the HMBC experiment and the hydrogen connected to this carbon was observed at 2.32 ppm. Furthermore, these two hydrogens again showed HMBC correlations with the very weak signal carbonyl carbon (C-1). On the other hand, the signals that have not been explained so far have six double bond signals, four methylene and terminal methyl groups, and when judged by their carbon chemical shift values, it can be assumed that the double bonds of methylene are arranged in a linear structure And was confirmed from COZY data. These linear partial structures were identified from the COZY correlation with hydrogen at 3.54 ppm (H-11) and 2.18 ppm (H-12), which is linked to carbon number 11. And when the double bond consisting of carbon and 8 9 has been determined to be E form from these carbon pairs of the hydrogen attached to the coupling constant (J = 15.7 Hz) remaining four double bonds is to determine the chemical shift of carbon Z . Thus, the structure of Compound 1 was found to be (4 Z , 8 E , 13 Z , 16 Z , 19 Z ) -7,10,11-trihydroxydocosa-4,8,13,16,19-pentaenoic acid, The structure is a new structure not known until now. MS / MS mass spectrometry of this structure revealed that the m / z values were measured as 137, 149, 167, 205, and 223, and these molecular fragments could be interpreted from the structure of compound 1 (FIG. Table 1 summarizes the hydrogen and carbon NMR data for Compound 1 .

화합물 1에 대한 탄소 및 수소의 NMR 데이터NMR data of carbon and hydrogen for compound 1 nono Carbon δC Carbon δ C δC, multiplicityδ C, multiplicity 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22One
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22 179.3, C
35.6, CH2
24.4, CH2
131.2, CH
127.5, CH
36.3, CH2
73.0, CH
136.2, CH
130.9, CH
76.0, CH
75.9, CH
31.8, CH2
129.4, CH
130.7, CH
26.8, CH2
129.0, CH
129.5, CH
26.4, CH2
128.2, CH
132.8, CH
21.5, CH2
14.7, CH3 179.3, C
35.6, CH 2
24.4, CH 2
131.2, CH
127.5, CH
36.3, CH 2
73.0, CH
136.2, CH
130.9, CH
76.0, CH
75.9, CH
31.8, CH 2
129.4, CH
130.7, CH
26.8, CH 2
129.0, CH
129.5, CH
26.4, CH 2
128.2, CH
132.8, CH
21.5, CH 2
14.7, CH 3
2.32, m
2.33, m
5.48, m
5.48, m
2.32, m
4.10, q (6.1)
5.74, dd (15.7, 6.1)
5.76, dd (15.7, 4.7)
3.96, t (4.7)
3.54, dt(7.8, 4.7)
2.18, dt (14.9, 7.8); 2.34, m
5.37, m
5.43, m
2.85, t (5.9)
5.34, m
5.34, m
2.81, t (5.9)
5.29, m
5.36, m
2.08, dq (7.6, 7.6)
0.96, t (7.6)
2.32, m
2.33, m
5.48, m
5.48, m
2.32, m
4.10, q (6.1)
5.74, dd (15.7, 6.1)
5.76, dd (15.7, 4.7)
3.96, t (4.7)
3.54, dt (7.8, 4.7)
2.18, dt (14.9, 7.8); 2.34, m
5.37, m
5.43, m
2.85, t (5.9)
5.34, m
5.34, m
2.81, t (5.9)
5.29, m
5.36, m
2.08, dq (7.6, 7.6)
0.96, t (7.6)

실험결과 2. Results 2. 화합물compound 22 의 구조 결정Structure determination

화합물 2의 구조는 질량분석과 NMR 스펙트럼으로부터 결정할 수 있었다. 먼저 화합물 2의 분자량은 ESI-LRMS에서 측정한 결과 [M + Na]+ m/z = 889와 [M - H]- m/z = 866값이 주어졌으며 이로부터 화합물 2의 분자량은 867로 결정되었다. 수소 NMR스펙트럼에서는 고자장 영역에서 삼중선 형태의 신호가 두 개 나타났으며 1.28 ppm에 큰 신호와 함께 저자장 영역 5.35 ppm 부근에 나타난 특징적인 수소선들로부터 고도 불포화 탄화수소가 포함되어 있다는 사실을 확인할 수 있었다. 이 외 화합물 2의 수소 스펙트럼에서 또 다른 특징은 4.75 ppm에 이중선으로 주어진 신호와 함께 2.6 ~ 4.5 ppm영역에 잘 분리된 NMR신호들이 관찰되었다 [도 6]. 탄소 스펙트럼에서는 174.2 과 175.1 ppm에 카보닐 작용기에 해당되는 두 개의 신호가 관찰되었으며 또한 불포화 탄화수소에 해당되는128 ~ 132 ppm영역과 26.6 ~ 26.5 ppm 에 여러 개의 탄소신호가 중첩되어 나타났다 [도 7]. HSQC 스펙트럼으로부터 탄소와 연결된 수소 원자들을 지정할 수 있었으며 COSY 스펙트럼을 바탕으로 수소들간의 연결성을 나타낼 수 있었다. The structure of compound 2 could be determined from mass spectrometry and NMR spectrum. First, the molecular weight of compound 2 was measured in the ESI-LRMS [M + Na] + m / z = 889 and [M - H] - m / z = the 866 value was given molecular weight of from which Compound 2 is determined to be 867 . In the hydrogen NMR spectrum, two triplet signals appeared in the high-field region, and a large signal at 1.28 ppm and a hyper-unsaturated hydrocarbon from the hydrogen lines near the 5.35 ppm authorship region there was. Another feature in the hydrogen spectrum of the other Compound 2 was the well-separated NMR signals in the 2.6 to 4.5 ppm region with a signal given in a double line at 4.75 ppm (Fig. 6). In the carbon spectrum, two signals corresponding to carbonyl functional groups were observed at 174.2 and 175.1 ppm, and several carbon signals were superimposed on the 128 to 132 ppm region corresponding to the unsaturated hydrocarbon and 26.6 to 26.5 ppm (Fig. 7). From the HSQC spectrum, the hydrogen atoms connected to the carbon could be specified and the connectivity between the hydrogen atoms could be shown based on the COZY spectrum.

먼저 COSY NMR 스펙트럼에서는 5.30 ppm에 있는 H-2는 3.57 과 4.11 ppm 에 있는 H-1과 상호 연결성을 보여주었고 더 나아가 4.18 과 4.49 ppm에 있는 H-3번과도 연결성을 보여주었다. 이 연결은 탄소의 화학적 이동값으로부터 판단할 때 이들의 부분 구조는 glycerol 골격을 이루고 있다는 것을 알 수 있었다. 또한 4.75 ppm에 위치한 H-1'는 3.39 ppm 에 있는 H-2', H-2'는 H-3' 순차적으로 H-4', H-5'와 상호 연결을 보여주었으며 H-5'는 2.91 과 3.34 ppm에 있는 H-6'과 연결을 보여 주었다 [도 8]. 이와 함께 H-1'는 69.9 ppm 에 있는 C-5'와 HMBC연결관계를 나타내었는데 이는 6탄당의 골격을 제시하였다. 주어진 6탄당에서 특이한 부분은 6번 위치에 있는 탄소의 화학적 이동값이 54.3 ppm으로 관찰되었다. 일반적인 6탄당의 6번 탄소의 화학적 이동값인 약 65.0 ppm과 비교해 볼 때 화합물 2에서는 고자장의 값으로 이동된 것을 알 수 있다. 문헌값과 비교한 결과 6번 탄소에 sulfonate 작용기가 붙어 있는 sufoquinovose로 추정되었으며 이는 IR 스펙트럼에서 1168 와 1034 cm-1 (S=O 작용기에 해당)에 나타난 신호와 질량분석 데이터에서 나타난 파편값 m/z = 663 (분자량 차이 226)으로부터 이를 증명할 수 있었다. 구조가 밝혀진 6 탄당은 앞서 설명된 glycerol의 1번 위치에 연결되어 있으며 이에 대한 증거로는 glycerol내에 있는 3.57과 4.11 ppm (H-1)의 수소가 100.1 ppm에 있는 anomeric 탄소(C-1')와 HMBC 상관관계가 있었다. 지금까지 NMR 데이터에서 해석되지 않은 신호들은 두 개의 서로 다른 지방산에 해당되었다. 이에 대한 근거로 negative MS/MS 질량 분석 데이터는 m/z 537과 609 파편 조각에 해당되는 분자량이 보여주었는데 이에 해당되는 두 지방산은 DHA(docosahexaenoic acid)와 palmitic acid로 주어졌다 [도 9]. Glycerol 골격에 두 지방산의 위치는 HMBC 스펙트럼으로부터 결정되었다. 먼저 palmitic acid에 있는 H-3˝′ (2.31 ppm)과 glycerol 에 있는 H-3 (4.18 ppm)이 공통으로 175.1 ppm의 탄소와 HMBC 상관성을 보여 주었으며 DHA분자에 있는 H-2˝ (2.38 ppm)의 신호가 174.2 ppm에 있는 다른 카보닐 탄소와 HMBC상관성을 보여주었다. 이로부터 glycerol의 2번 위치에 DHA가 3번 위치에 palmitic acid이 연결되어 있는 것을 알 수 있었다. 따라서 결정된 화합물 2의 구조는 sulfoquinovosyl diacylglycerol 계열의 물질이며 특히 glycerol 2번 위치에 DHA가 연결된 화합물은 지금까지 알려지지 않은 신규 구조이다. First, in the COSY NMR spectrum, H-2 at 5.30 ppm showed interconnection with H-1 at 3.57 and 4.11 ppm, and also with H-3 at 4.18 and 4.49 ppm. This linkage can be seen from the chemical shift values of carbon that their partial structure is glycerol skeleton. H-1 'at 4.75 ppm showed interconnection with H-4' and H-5 'at H-2' and H-3 'at 3.39 ppm and H-5' 2.91 and 3.34 ppm (Fig. 8). In addition, H-1 'showed a linkage between C-5' at 69.9 ppm and HMBC, suggesting the skeleton of hexose. The chemical shift of the carbon at position 6 was found to be 54.3 ppm. Compared to the chemical shift value of about 65.0 ppm, which is the general chemical shift of 6-carbon of hexose, it can be seen that compound 2 has shifted to the value of hysteresis. As a result of comparison with literature values, it was estimated to be sufoquinovose with sulfonate functional group at carbon number 6, which is the signal at 1168 and 1034 cm -1 (corresponding to the S = O functional group) in the IR spectrum and the fragment value m / z = 663 (molecular weight difference 226). The hexose structure revealed is linked to position 1 of the glycerol as described above and the anomeric carbon (C-1 ') at 100.1 ppm of hydrogen at 3.57 and 4.11 ppm (H-1) in glycerol, And HMBC correlations. So far, uninterpreted signals in the NMR data corresponded to two different fatty acids. As a basis for this, negative MS / MS mass spectrometry data showed molecular weights corresponding to fragments of m / z 537 and 609, the corresponding two fatty acids being DHA (docosahexaenoic acid) and palmitic acid (Fig. The position of the two fatty acids in the glycerol skeleton was determined from the HMBC spectrum. First, H-3 '(2.31 ppm) in palmitic acid and H-3 (4.18 ppm) in glycerol were common, showing 175.1 ppm carbon and HMBC correlation, and H-2 " Showed HMBC correlations with other carbonyl carbons at 174.2 ppm. From this, it was found that palmitic acid was linked to glycerol at position 2 and DHA at position 3. The structure of compound 2 thus determined is a sulfoquinovosyl diacylglycerol family compound, in particular, a compound in which DHA is linked to glycerol position 2 is a novel structure not known until now.

화합물 2에 있는 sulfoguinovose는 anomeric 수소 (H-1˝)의 짝결합 상수값 (J = 3.9 Hz)으로 부터 α anomer로 결정되었으며 또한 H-2˝ ~ H-5˝ 의 짝결합 상수값으로 부터 이들의 상대적인 입체 구조를 정할 수 있었다. Sufoquniovose와 glycerol에 있는 chiral 탄소에 대한 절대 입체 구조는 결정하지 않았지만 유사한 분자 구조를 가진 화합물과의 optical rotation 값 ([α] 34.8, c=0.1)의 비교로부터 측정값으로부터 화합물 2의 구조는 1-(1′-O-α-D-sulfoquinovosyl)-2-(4˝Z, 7˝Z, 10˝Z, 13˝Z, 16˝Z, 19˝Z-docosahexaenyl)-3-palmitosyl-syn-glycerol로 결정되었다. 표 2는 화합물 2에 대한 수소와 탄소 NMR 데이터를 정리한 것이다.The sulfoguinovose in compound 2 was determined as an anomer from the mating constant ( J = 3.9 Hz) of the anomeric hydrogen (H-1 "), and from the mating constant values of H-2˝ to H-5˝ The relative three-dimensional structure of the microstructure was determined. The absolute stereostructure of chiral carbon in Sufoquniovose and glycerol was not determined, but from the comparison of the optical rotation value ([α] 34.8, c = 0.1) with the compound with similar molecular structure, the structure of compound 2 from 1- (1'- O -α-D-sulfoquinovosyl ) -2- (4˝ Z, 7˝ Z, 10˝ Z, 13˝ Z, 16˝ Z, 19˝ Z -docosahexaenyl) -3-palmitosyl- syn -glycerol Respectively. Table 2 summarizes hydrogen and carbon NMR data for compound 2. < tb >< TABLE >

화합물 2에 대한 탄소 및 수소의 NMR 데이터 NMR data of carbon and hydrogen for compound 2 nono Carbon δC Carbon δ C δC , multiplicityδ C , multiplicity 1
2
3
1'
2'
3'
4'
5'
6'
1"
2"
3"
4",5",7",8",10",11",
13",14",16",17",19"
6", 9",12",15",18"
20"
21"
22"
1"'
2"'
3"'
4"' ~ 15"'
16"'One
2
3
One'
2'
3 '
4'
5 '
6 '
One"
2"
3 "
4 ", 5 ", 7 ", 8 ", 10 &
13 ", 14", 16 ", 17", 19 "
6 ", 9 ", 12 ", 15 ", 18 "
20 "
21 "
22 "
One"'
2"'
3 "'
4 "'~ 15"'
16 "' 67.1, CH2
71.9, CH
64.4, CH2
100.1, CH
73.5, CH
74.9, CH
75.1, CH
69.9, CH
54.3, CH2
174.2, C
35.1, CH2
23.8, CH2
128.2 ~ 129.5, CH

26.6 ~ 26.5, CH2
132.8, CH2
21.6, CH2
14.7, CH3
175.1, C
35.0, CH2
26.0, CH2
30.8 ~ 30.3, CH2
14.5, CH3 67.1, CH 2
71.9, CH
64.4, CH 2
100.1, CH
73.5, CH
74.9, CH
75.1, CH
69.9, CH
54.3, CH 2
174.2, C
35.1, CH 2
23.8, CH 2
128.2 to 129.5, CH

26.6 to 26.5, CH 2
132.8, CH 2
21.6, CH 2
14.7, CH 3
175.1, C
35.0, CH 2
26.0, CH 2
30.8 to 30.3, CH 2
14.5, CH 3 3.57, dd (10.8, 6.4); 4.11, dd (10.8, 5.1)
5.30, m
4.18, dd (12.0, 6.9); 4.49, dd (12.0, 2.9)
4.75, d (3.9)
3.39, dd (9.5, 3.9)
3.62, t (9.5)
3.08, dd (9.5, 9.0)
4.07, td (9.0, 2.0)
2.91, dd (14.2, 9.0); 3.34, dd (14.2, 2.0)

2.38, m
2.38, m
5.39 ~ 5.30, m

2.87 ~ 2.80, m
5.36, t (5.9)
2.08, quint (7.6)
0.97, t (7.6)

2.31, t (7.6)
1.58, m
1.28, m
0.89, t (7.1)3.57, dd (10.8, 6.4); 4.11, dd (10.8, 5.1)
5.30, m
4.18, dd (12.0, 6.9); 4.49, dd (12.0, 2.9)
4.75, d (3.9)
3.39, dd (9.5, 3.9)
3.62, t (9.5)
3.08, dd (9.5, 9.0)
4.07, td (9.0, 2.0)
2.91, dd (14.2, 9.0); 3.34, dd (14.2, 2.0)

2.38, m
2.38, m
5.39 to 5.30, m

2.87 to 2.80, m
5.36, t (5.9)
2.08, quint (7.6)
0.97, t (7.6)

2.31, t (7.6)
1.58, m
1.28, m
0.89, t (7.1)

3. 생리활성 결과3. Physiological activity results

RAW 264.7 대식세포에서 화합물의 NO 생성 저해 효과를 알아보기 위하여 LPS 자극을 준 세포에 화합물 1을 0.1, 1, 5, 10 μM를 처리하여 24시간 후의 NO 함량을 측정하였다. LPS로 활성화된 RAW264.7 대식세포에서 NO의 생성량이 크게 증가하였으며, 화합물을 처리한 경우 전체 투여농도에서 농도의존적으로 NO 생성량이 감소하였다[도 6]. MTT assay 결과 모든 투여 농도에서 세포독성은 전혀 관찰되지 않았다.In order to investigate the inhibitory effect of compounds on the production of NO in RAW 264.7 macrophages, the NO content of LPS-stimulated cells was treated with Compound 1 at 0.1, 1, 5, and 10 μM for 24 hours. NO production was significantly increased in RAW264.7 macrophages activated with LPS, and when the compounds were treated, the NO production was decreased in a concentration-dependent manner at the total dose (Fig. 6). MTT assay showed no cytotoxicity at all dose levels.

토의discussion

와편모조류 Oxyrrhis marina는 지방산이 풍부하였으며 그 중 특히 DHA함량이 다른 미세조류에 비하여 높은 편이었다. 이 종을 대량 배양하여 새로운 화합물을 찾는 연구를 수행한 결과 메탄올 추출 용액에서 DHA로부터 유도된 신규 화합물 2종을 발견하였다. 화합물 1은 DHA분자가 산화되어 생긴 oxylipin계이며 이는 기존 EPA(eicosapentaenoic acid)로 부터 유도된 많은 oxylipin과 차별되며 또한 DHA로부터 산화된 기존 화합물과도 차별되었다. 함께 분리된 화합물 2는 DHA분자가 붙은 sulfolipid 화합물이다. 이 화합물은 glycerol 골격에서 1번 위치에 sulfoquinovose, 2번 위치에 DHA, 3번 위치에 palmitic acid가 연결되어 있다. sulfolipid에 대한 많은 문헌과 특허가 있지만 현재 분리된 화합물 2는 DHA가 결합되어 있어 차별성이 있는 신규 구조이다. Oxyrrhis marina was rich in fatty acids and DHA content was higher than other microalgae. We have found two new compounds derived from DHA in a methanol extract solution. Compound 1 was an oxylipin system formed by oxidation of DHA molecules, which was differentiated from many oxylipins derived from EPA (eicosapentaenoic acid) and also differentiated from existing compounds oxidized from DHA. Compound 2, isolated together, is a sulfolipid compound with a DHA molecule attached. This compound has sulfoquinovose at position 1, DHA at position 2, and palmitic acid at position 3 in the glycerol backbone. There are many documents and patents on sulfolipid, but currently isolated compound 2 is a new structure with differentiation due to the binding of DHA.

위 두 화합물 모두 세포독성이 없으며 기존 유사화합물에서 다양한 생리활성을 보여 주고 있기 때문에 이들 화합물도 뛰어난 활성을 보여줄 것으로 기대된다. Both of these compounds are not cytotoxic and exhibit a variety of physiological activities in similar compounds, and these compounds are expected to exhibit excellent activity.

Claims (2)

하기 화학식 1로 나타나는 신규의 oxylipin 계열의 화합물.
[화학식 1]
Figure pat00005

A novel oxylipin-based compound represented by the following formula (1).
[Chemical Formula 1]
Figure pat00005

 하기 화학식 1로 나타나는 신규의 DHA 유도체 화합물.
[화학식 2]
Figure pat00006
A novel DHA derivative compound represented by the following formula (1).
(2)
Figure pat00006
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