[논문]감태에서 분리된 플로로탄닌의 염증 및 알러지 억제 효과

이승수; 방면호; 전형주; 황태규; 양선아

doi:10.5352/jls.2018.28.10.1170

감태에서 분리된 플로로탄닌의 염증 및 알러지 억제 효과
Anti-inflammatory and Anti-allergic Effects of Phlorofucofuroeckol A and Dieckol Isolated from Ecklonia cava 원문보기

생명과학회지 = Journal of life science, v.28 no.10 = no.222, 2018년, pp.1170 - 1178

이승수 (경희대학교 피부생명공학센터) , 방면호 (경희대학교 피부생명공학센터) , 전형주 (장안대학교 식품영양학과) , 황태규 (우석대학교 호텔관광학과) , 양선아 (계명대학교 식품가공학과)

초록
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갈조류인 감태(Ecklonia cava)의 에틸아세테이트 분획으로부터 phloroglucinol, eckol, eckstolonol, triphlorethol-A, phlorofucofuroeckol A, dieckol의 6종의 phlorotannin이 분리되었으며, NMR 및 MS분석으로 구조가 규명되었다. 감태의 주요 활성물질 분석을 위하여, 분리된 6종 물질에 대한 항산화, 항염증 및 항알러지 효과를 평가하였다. 그 중 phlorofucofuroeckol A와 triphlorethol-A가 라디컬(DPPH, ABTS) 소거 활성이 높았으며, 염증 반응에 대한 활성을 분석하기 위하여 LPS를 처리한 대식세포주인 Raw264.7에서 산화질소(NO) 생성 억제활성을 측정한 결과, phlorofucofuroeckol A의 NO 생성 억제 효과가 가장 큰 것으로 나타났다. 또한, 알러지 반응에 대한 억제활성을 분석하기 위하여 IgE-항체로 활성화시킨 비만세포주 RBL-2H3에서 분비되는 ${\beta}-Hexosaminidase$를 측정한 결과, dieckol이 농도의존적으로 가장 높은 억제 효과를 나타냈다. 따라서, 본 연구에서 항염증 및 항알러지 활성을 중심으로 분리된 6종의 플로로탄닌 중 phlorofucofuroeckol A와 dieckol은 감태의 알러지 염증 억제활성을 나타내는 주요 물질로 사료된다.

Abstract ▼ AI-Helper

The anti-inflammatory effect of brown alga Ecklonia cava is well known, and several phlorotannins have also been reported. In this study, major active components for anti-allergy and anti-inflammation were identified by NMR and MS analysis, and the levels of effectiveness were compared. Six major phlorotannins-phloroglucinol, eckol, eckstolonol, triphlorethol-A, phlorofucofuroeckol A, and dieckol-were isolated from the ethyl acetate fraction of E. cava. In order to analyze the major active substances in E. cava, antioxidant, anti-inflammatory, and anti-allergic effects were evaluated for the six separate substances. Antioxidant capacities of each phlorotannin were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, where phlorofucofuroeckol A and triphlorethol-A had the highest radical scavenging capacity in respective radical scavenging assays. Phlorofucofuroeckol A exhibited the highest inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells among phlorotannins tested. Dieckol inhibited the release of ${\beta}-hexosaminidase$, a marker for the release of histamine in mast cells, in a dose-dependent manner in antigen-stimulated RBL-2H3 mast cells. Additionally, no cytotoxicities were observed at 1 and $2{\mu}g/ml$ in both phlorofucofuroeckol A and dieckol. These results suggest that phlorofucofuroeckol A and dieckol may play a key role in allergic inflammatory reactions.

주제어

표/그림 (6)

그림 Fig. 1. Chemical structures of phlorotannins isolated from Ecklonia cava. (1) phloroglucinol, (2) eckol, (3) eckstolonol, (4) triphlor-ethol-A, (5) phlorofucofuroeckol A, (6) dieckol.
표 Table 1. Identification of six phlorotannins in Ecklonia cava and data from FAB-MS, ¹H-NMR and ¹³C-NMR
그림 Fig. 2. Effects of the six phlorotannins from Ecklonia cava on LPS-induced NO production in RAW 264.7 cells. Nitrite levels were measured in the supernatants. Data are represented as the means±SD of three independent experiments. Values with different superscript letters are significantly different (p<0.05). The asterisk (*) represents significantly different from control (p<0.05). L-NAME used as a positive control at a concentration of 250 μM.
그림 Fig. 3. Inhibitory effects of the six phlorotannins from Ecklonia cava on β-hexosaminidase release in RBL-2H3 cells induced by IgE with DNP-BSA. The levels of released β-hexosaminidase were measured in the supernatants. Data are represented as the means±SD of three independent experiments. Values with different superscript letters are significantly different (p<0.05). The asterisk (*) represents significantly different from control (p<0.05). Wortmannin was used as a positive control at a concentration of 500 nM.
표 Table 2. Antioxidant capacities of six phlorotannins found in Ecklonia cava¹⁾
그림 Fig. 4. Cytotoxicity and inhibitory effects of dieckol on β-hexosaminidase release from RBL-2H3 cells induced by IgE with DNP-BSA. Letters on bars indicate significant differences according to Duncan's multiple range test (p<0.05, n=3).

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제안 방법

MPLC (TELEDYNE Isco Inc, Lincoln, NE, USA) and preparative HPLC (Waters, MA, USA) were used for separation. TLC analysis was performed using Kiesel gel 60 F₂₅₄ and RP-18 F_254S (Merck, Darmstradt, Germany) resins and detected using a UV lamp Spectroline Model ENF-240 C/F (Spectronics Corporation, New York, NY, USA) and 10% H₂SO₄ solution. FAB-MS was recorded on a JEOL JMSAX700 (Tokyo, Japan).
The objectives of the present study were to isolate and identify major phlorotannins of E. cava, to evaluate the levels of antioxidant capacity of each phlorotannins, and ultimately to investigate whether major phlorotannins showed the anti-inflammatory and anti-allergic effects.
To determine the cytotoxicity of the phlorotannins, cell viability was determined using the MTT assay, in which ac-tive mitochondria reduce MTT into formazan dye. RAW 264.

대상 데이터

cava was collected in July, 2011 in Jeju, Korea and identified by Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Korea. A voucher specimen (KHU 0111107) was deposited at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.
E. cava was collected in July, 2011 in Jeju, Korea and identified by Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Korea. A voucher specimen (KHU 0111107) was deposited at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.
RAW 264.7 cells, a murine macrophage cell line, and RBL-2H3 cells, a rat basophilic leukemia cell line, were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). RAW 264.

데이터처리

Letters on bars indicate significant differences according to Duncan’s multiple range test (p<0.05, n=3).
Data are expressed as the means ± standard deviation (SD). One way ANOVA test (using SPSS 20 statistical software) was used to compare the mean values of each treatment. Significant differences were verified by Duncan’s multiple range tests (p<0.
Significant differences were verified by Duncan’s multiple range tests (p<0.05).

성능/효과

In conclusion, six phlorotannins obtained from a brown alga, E. cava included phloroglucinol, eckol, eckstolonol, triphlorethol-A, phlorofucofuroeckol A, and dieckol, which identified by MS and NMR analyses. Antioxidant capacities of six phlorotannins were ranked by the DPPH and ABTS assays, where phlorofucofuroeckol A and triphlorethol-A had the highest antioxidant capacity in respective assays.
4% at 10 μg/ml without affecting cell viability of RBL-2H3 cells. The other five phlorotannins tested in this study showed less inhibitory effects with approximately 20.5-37.8% inhibition. We tested cell viability using the MTT assay to ensure that the decrease in NO production was not due to cell death.

후속연구

cava. Thus, research is still needed to investigate which phlorotannins from E. cava has strong effects on anti-inflammatory and anti-allergic activities. The objectives of the present study were to isolate and identify major phlorotannins of E.

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