RAW 264.7 세포내 지질다당류로 유도된 산화질소의 생성 억제에 따른 대복피 유래 페놀성 화합물의 분리

학위논문 (석사)-- 서울대학교 대학원 : 약학과(천연물과학전공), 2015. 8. 김영식.Arecae Pericarpium는 Areca catechu L. (Arecaceae)의 과피이다. 중의학에서는 복부 팽만, 구토증세, 설사를 완화하는 곽향정기산에, 한의학에서는 혈관질환의 치료를 위한 가미정기산에 처방된다. Arecae Pericarpium의 항산화 및 항진균 효과와 그것의 지표물질 추출이 보고된 바 있다. 그러나 Arecae Pericarpium의 항염증 효과는 현재까지 보고된 바가 없어 본 연구에서는 RAW 264.7 대식세포에서 산화질소 생성을 억제하는 Arecae Pericarpium의 항염성분 분리를 목표로 하였다. Arecae Pericarpium 유래 물질의 분리는 RAW 264.7 대식세포에서 LPS 처리에 의해 유도되는 산화질소 생성의 억제 정도에 따라 수행되었다. 건조된 Arecae Pericarpium을 메탄올로 침출하여 농축하고 헥산, 메틸렌클로라이드, 에틸아세테이트, 부탄올을 이용하여 순차적 용매 분획을 시행하였다. 산화질소 생성 억제 활성이 뛰어난 메틸렌클로라이드와 에틸아세테이트 층을 선택하여 물질 분리를 진행하였다. 메틸렌클로라이드 층은 실리카겔 컬럼크로마토그래피를 통하여 7개의 분획(MC1A~MC1G)으로 나누었다. 그 중 MC1A, MC1B, MC1G 분획이 산화질소 생성 억제 효과를 보였으며 MC1G은 syringol dimer (compound I)를 단일 물질로 포함하고 있음을 밝혔다. Diaion® HP-20 수지를 이용한 컬럼크로마토그래피를 통한 MC1A 유래의 20 %, 30 % 메탄올 용출액에서 catechol (compound II)을 정제하였다. MC1B에서 유래한 4-hydroxybenzaldehyde (compound III), vanillin (compound IV), 4-hydroxyacetophenone (compound V), apocynin (compound VI) 등 네 개의 물질은 헥산-에틸아세테이트-메탄올-물(2:5:1:4, v/v/v/v)로 구성된 용매계를 이용한 HPCCC(고성능향류크로마토그래피)와 preparative HPLC를 통하여 분리되었다. Ethyl acetate 층은 Diaion® HP-20 수지를 이용한 컬럼크로마토그래피를 통하여 분획하고 그 중 20% 메탄올 용출액을 선택하여 HPCCC를 수행하였다. 헥산-에틸아세테이트-메탄올-물 2:5:2:5(v/v/v/v)의 비율로 조성된 용매계를 통하여 protocatechuic acid (compound VII)와 4-hydroxybenzoic acid (compound VIII)를 분리하였다. 본 연구에서 분리된 Arecae Pericarpium 유래 화합물들이 중에서 catechol이 LPS에 의해 유도되는 산화질소 생성 억제의 주된 활성에 기여함을 밝혔다.Isolation of Phenolic Compounds from Arecae Pericarpium Guided by Inhibition of LPS-Induced Nitric Oxide Production in RAW 264.7 Cells RAW 264.7 세포내 지질다당류로 유도된 산화질소의 생성 억제에 따른 대복피 유래 페놀성 화합물의 분리 August 2015 Amelia Indriana Natural Product Science Major College of Pharmacy Master Course in Graduate School Seoul National University Abstract Isolation of Phenolic Compounds from Arecae Pericarpium Guided by Inhibition of LPS-Induced Nitric Oxide Production in RAW 264.7 Cells Amelia Indriana Natural Products Science Major College of Pharmacy Master Course in the Graduate School Seoul National University Arecae Pericarpium is the nut husk of Areca catechu L. (Arecaceae). In Traditional Chinese Medicine, it is used in Huo Xiang Zheng Qi formula to cure abdominal distension, vomiting, and diarrhea and Gami-Jeonggisan formula in Traditional Korean Medicine to treat vascular diseases. Arecae Pericarpium has been studied for its antioxidative activity, antifungal activity, and phytochemical compounds isolation. However, its antiinflammatory activity has not been reported yet. This study aimed to isolate compounds from Arecae Pericarpium guided by the inhibition of nitric oxide production on RAW 264.7 macrophage cells. Dried Arecae Pericarpium was macerated in methanol. The macerate was evaporated, dissolved in 10% methanol and partitioned with hexane, methylene chloride, ethyl acetae and n-butanol. Methylene chloride and ethyl acetate fraction, which inhibited nitric oxide production to lower levels compared to other fractions, were further separated. Methylene chloride fraction was subjected to repeated silica gel column chromatography to obtain seven fractions (MC1A to MC1G fraction). MC1A, MC1B and MC1G fractions inhibited the LPS-induced nitric oxide production. MC1G was obtained as single compound and identified as syringol dimer (compound I). MC1A was subjected through Diaion® HP-20 column chromatography and catechol (compound II) was obtained from 20% and 30% MeOH column fraction. Four compounds, 4-hydroxybenzaldehyde (compound III), vanillin (compound IV), 4-hydroxyacetophenone (compound V) and apocynin (compound VI), were isolated from MC1B fraction by combining HPCCC (hexane-ethyl acetate-methanol-water system 2:5:1:4 v/v) and preparative HPLC. Ethyl acetate fraction was subjected to Diaion® HP-20 column chromatography and 20% MeOH column fraction was separated using high performance counter-current chromatography (hexane-ethyl acetate-methanol- water system 2:5:2:5 v/v) to obtain protocatechuic acid (compound VII) and 4-hydroxybenzoic acid (compound VIII). All isolated compounds were examined and the result suggested that catechol might be the primary contributing active compound to the suppression of LPS-induced NO production by Arecae Pericarpium.TABLE OF CONTENTS I. INTRODUCTION 1 1. Bioassay-guided Isolation 1 2. Inflammation 2 3. Inducible Nitric Oxide Synthase and Nitric Oxide 3 4. High Performance Counter-current Chromatography (HPCCC) 4 5. Areca catechu L. 5 6. Purpose of This Study 8 II. MATERIALS AND METHODS 9 1. MATERIALS 9 1.1 Plant Materials 9 1.2 Chemicals and Reagents 9 1.3 Instruments 10 2. METHODS 12 2.1 Cell Culture 12 2.2 Griess Reagent Nitrite Assay 12 2.3 Cell Viability 13 2.4 Determination of PGE2 detection 13 2.5 NF-ĸB Secretory Alkaline Phosphatase Reporter Gene Assay 14 2.6 Extraction 14 2.7 Silica column chromatography 15 2.8 Diaion® HP-20 resin column chromatography 16 2.9 Selection of the two-phase solvent system and measurement of the partition coeffient (K) and settling times 16 2.10 HPCCC Separation 17 2.11 Preparative HPLC Separation 17 2.12 Identification of isolated compounds 18 III. RESULTS 20 1. Preliminary study 20 2. Separation of methylene chloride fraction 22 3. Suppression of LPS-induced NO production and cell viability assay by silica CC fractions from methylene chloride fraction 24 4. Separation of MC1 fraction 26 5. Suppression of MC1A ? MC1G fraction on LPS-induced NO production in RAW 264.7 macrophages 28 6. Separation of MC1B fraction 30 7. Separation of Ethyl Acetate Fraction 35 8. Suppression of LPS-induced NO production by ethyl acetate fractions obtained from Diaion® HP-20 resin column chromatography separation 39 9. Structure Identification of Isolated Compounds 41 9.1 Identification of compound I 41 9.2 Identification of compound II 45 9.3 Identification of compound III 49 9.4 Identification of compound IV and V 54 9.5 Identification of compound VI 59 9.6 Identification of compound VII 65 9.7 Identification of compound VIII 70 10. Suppression of LPS-induced NO activity of isolated compounds 76 IV. DISCUSSION 78 V. CONCLUSION 81 REFERENCES 82 ABSTRACT IN KOREAN 86Maste