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Anti-cancer and anti-inflammatory effects of eutigosides from eurya emarginata

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Alternative Title
우묵사스레피의 eutigosides에 의한 항암 및 항염증 효과
Abstract
우묵사스레피 (Eurya emarginata)는 차나무과의 상록 관목수로 제주도 같은 따뜻한
도서지방의 바닷가 산지에서 주로 자생하며, 특히 건조, 해풍, 먼지, 대기오염 등 각
종 공해에 강하다. 지역적으로 중국, 대만, 일본, 인도 등에 분포하며, 열매가 쥐똥 같
고 해변에 자생한다하여 섬쥐똥나무라 불리워지기도 하며 사스레피나무와 생김새가
비숫하나 잎끝이 뒤로 말리어 우묵하게 들어가서 우묵사스레피라 한다.
민간에서는 그 잎이 이뇨와 담 제거 및 종기제거 등의 약재로 쓰이고 있으나, 그
잎의 유효성분이나 생리활성에 대한 보고는 거의 없는 실정이다. 이에 본 연구자는
우묵사스레피 잎에서의 유효활성 성분을 탐색하였고, 또한 유효활성 성분들이 항암
및 항염증 효과에 있어 어떤 작용을 하는지를 알아보았다.
최근 apoptosis가 세포생물학의 주요 주제로 떠오르면서 chemotherapeutic 및
chemopreventive 작용기전 연구로 apoptosis에 대한 많은 연구가 이루어지고 있다. 이
에 우묵사스레피 잎에서 분리된 활성성분 중 Eutigoside C를 HL-60 혈액종양암 세포
를 이용하여 세포증식억제률, DNA fragmentation, morphologic changes, sub-G1
hypodiploid cells를 관찰한 결과 Eutigoside C에 의해 apoptosis가 유도됨을 확인할 수 있었
다. 그리고 Westren blot과 RT-PCR을 통한 apoptosis 관련 유전자 및 세포주기 관련 유전자
변화 등을 통해 유의한 결과를 얻을 수 있었다. 또한, 우묵사스레피 잎에서 분리한
Eutigoside B와 C로부터 염증반응의 주체가 되는 대식세포 모델계에서 murine
macrophage cell line RAW264.7 으로부터의 LPS 자극에 의한 TNF-α, IL-6, IL-1β와 같
은 pro-inflammatory cytokine과 iNOS 및 COX-2의 발현 억제 효과를 관찰한 결과, TNF- ,
IL-6 형성억제 및 iNOS, COX-2의 발현억제, 그리고 높은 NO 형성억제 효과를 얻을
수 있었다. 이러한 결과는 우묵사스레피의 유효성분으로부터 암을 예방하거나 치료할
수 있는 항암제 또는 항염증제 성분의 분리 및 그 작용기전 연구에 중요한 기초 자
료가 될 것이라 사료된다.
Author(s)
박수영
Issued Date
2003
Awarded Date
2003.
Type
Dissertation
URI
https://oak.jejunu.ac.kr/handle/2020.oak/14632
Alternative Author(s)
Park, Soo Young
Affiliation
제주대학교 대학원
Department
대학원 생물학과
Advisor
김세재
Table Of Contents
CONTENTS ......................................................................................................ⅰ
LIST OF SCHEMES .........................................................................................ⅵ
LIST OF FIGURES ...........................................................................................ⅶ
LIST OF TABLES .............................................................................................ⅺ
OVERALL BACKGROUNDS ........................................................................1
PART ONE .......................................................................................................3
PART TWO .....................................................................................................59

PART ONE
ABSTRACT ........................................................................................................4
INTRODUCTION ..............................................................................................6
MATERIALS AND METHODS ...................................................................10
1. Purification of biologically active components from E. emarginata
1-1. Preparation of extracts from leaves of E. emarginata ..........................10
1-2. Characterization procedures ...........................................................................10
2. Analysis of cytotoxicity or apoptotic events
2-1. Cell cultures ...................................................................................................11
2-2. Cytotoxicity tests ...........................................................................................11
2-3. Cell surface antigen analysis ...........................................................................13
2-4. DNA fragmentation analysis .......................................................................13
2-5. Microscopic observation ...............................................................................14
2-6. Cell cycle phase distribution analysis .......................................................14
2-7. RNA extraction ..............................................................................................14
2-8. RT-PCR analysis ...........................................................................................15
2-9. Subcellular fractionation ................................................................................15
2-10. Western blot analysis .................................................................................16
RESULTS ..........................................................................................................20
1. Purification of biologically active components from E. emarginata
1-1. Extraction and Fractionation of E. emarginata leaves ..........................20
1-2. Isolation of the eutigoside B and C .................................................................22
2. Analysis of cytotoxic- or apoptotic events
2-1. Effects of the extracts of E. emarginata on the growth of tumor cells
.....................................................................................................................................28
2-2. Cell surface antigen analysis ............................................................................30
2-3. Effects of the EtOAc subfraction of E. emarginata on the growth of
HL-60 cells ..............................................................................................................30
2-4. Induction of apoptosis by E. emarginata in HL-60 cells ......................30
2-5. Expressions of c-Myc by the EtOAc fraction in HL-60 cells ..............36
2-6. Expressions of Bcl-2 and Bax by the eutigoside C in HL-60 cells ...........36
2-7. Release of mitochondrial cytochrome c into cytosol in HL-60 cells .......37
2-8. Degradation of PARP, an endogenous substrate of caspase-3 in HL-60 cells
.....................................................................................................................................37
3. Cell cycle regulation by the eutigoside C in HL-60 cells
3-1. Inhibition of cell growth by eutigoside C in HL-60 cells .........................45
3-2. Cell growth arrest induced by the eutigoside C from E. emarginata
.....................................................................................................................................45
DISCUSSION ....................................................................................................51

PART TWO
ABSTRACT .......................................................................................................60
INTRODUCTION .............................................................................................62
MATERIALS AND METHODS ...................................................................64
1. DPPH radical-scavenging activity ........................................................................64
2. Cell culture ..............................................................................................................65
3. Pro-inflarmmatory cytokines production in vitro ..............................................65
4. RNA extraction .......................................................................................................66
5. RT-PCR analysis ....................................................................................................66
6. Western blot analysis ............................................................................................68
7. Nitrite assay ...............................................................................................................69
RESULTS ...........................................................................................................70
1. Isolation of eutigosides from E. emarginata leaves and measurement of
DPPH radical-scavenging activity .......................................................................70
2. The effect of E. emarginata on LPS-induced IL-1β, IL-6, and TNF-α
secretion in RAW264.7 .........................................................................................71
3. TNF-α and IL-6 production in vitro ..................................................................76
4. Inhibition of LPS-mediated iNOS induction in RAW264.7 ...........................76
5. Inhibition of LPS-mediated COX-2 induction in RAW264.7 .........................81
6. Inhibition of LPS-mediated NO production in RAW264.7 ............................81

DISCUSSION ..................................................................................................88
LITERATURE CITED ....................................................................................93
ABSTRACT IN KOREAN .............................................................................110
Degree
Doctor
Publisher
제주대학교 대학원
Citation
박수영. (2003). Anti-cancer and anti-inflammatory effects of eutigosides from eurya emarginata
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General Graduate School > Biology
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