Anti-allergic effects of phlorotannins isolated from Ecklonia cava
- Alternative Title
- 감태로부터 phlorotannins 의 추출정제 및 그의 항알러지 효능
- Abstract
- 알러지는 천식, 아토피성 습진, 알러지성 비염, 결막염, 두드러기 등과 같은 질병을 유발하는 것으로 전체 인구의 약 20%정도가 가지고 있으며, 그 발병과 정도에 있어서 점차 심화되고 있는 추세이다. 최근 들어 신약 개발이 빠르게 진행되고 있음에도 불구하고, 현재까지 만족할 만한 항알러지 치료제는 없는 실정이다.
해조류는 다양한 생리활성을 나타내는 대사산물을 함유하고 있는 만큼 의약품으로서의 잠재적인 응용범위가 매우 넓다. 본 연구에서는 30여종의 해조류에서 높은 항알러지 활성을 나타내는 감태로부터 phloroglucinol (1), dieckol (2), 6,6’-bieckol (3), 그리고 1-(3’,5’-dihydroxyphenoxy)-7-(2”,4”,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (4)의 phlorotannin계 물질을 분리 정제하였다. 분리한 phlorotannin계 물질들이 호염기성 세포와 대식세포에 대한 세포독성을 나타내지 않는 것을 확인한 후 이들 세포를 이용하여 항알러지 효능을 조사하였다. 분리된 물질들 중 (2), (3), (4)는 calcium ionphore (A23187)와 IgE에 의하여 유도되는 basophil의 탈과립화를 저해하였다. 또한 분리된 phlorotannin계 물질은 호염기성 세포에서 발현되고 IgE친화력이 높은 receptor인 FcεRI의 유전자·단백질 발현실험을 통해 확인결과, receptor의 α-chain 발현을 저해함으로써 basophil의 탈과립화를 감소시키는 것을 확인할 수 있었다. 그리고 (2), (3)은 인간 대식세포(U937과 HL-60)의 histamine receptor와 histamine간의 상호작용에 대하여 농도의존적 경쟁적 저해현상을 나타내었다. 이러한 결과는 감태로부터 분리한 phlorotannin류들이 복합적인 항알러지 활성을 가지고 있고 향후 항알러지 기능성물질로서의 가능성을 제시하였다.
Marine algae yielded a large variety of bioactive metabolites, and some of them have biomedical potentials. In this study, marine algae were used for screening anti-allergic materials. Four phlorotannins, phloroglucinol (1), dieckol (2), 6,6′-bieckol (3), and 1-(3′,5′-dihydroxyphenoxy)-7-(2′′,4′′,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (4), were purified and characterized from marine brown alga, Ecklonia cava, which showed the strongest anti-allergic effects among 36 marine algae. Phlorotannins showed no adverse effect as tested by cytotoxicity assay using different cell lines. Anti-allergic actions of these compounds were evaluated using basophilic leukemia and macrophage. Among these compounds, compound 2 and 3 showed the strongest inhibitory effects, followed by compound 4, on degranulation of KU812F and RBL-2H3 cells, stimulated by calcium ionophore (A23187) and IgE. Studies on the gene and protein expression levels of FcεRI, the high affinity IgE receptor on basophils, revealed that these compounds also showed inhibitory effect on α chain expression of the receptor and therefore, decrease the degranulation of basophils. Moreover, the compounds exhibited anti-histaminic effects by inhibiting the binding between histamine and histamine receptors on human macrophage (U937 and HL-60) in dose-dependent manners, in which, compound 2 and 3 still showed the strongest inhibition. In all cases, compound 1 showed no anti-allergic effects. These results suggest that pholorotannins from Ecklonia cava have multiple anti-allergic potentials and can be used as anti-allergic materials after further evaluations.
- Issued Date
- 2008
- Awarded Date
- 2008. 8
- Type
- Dissertation
- Publisher
- 부경대학교 대학원
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Table Of Contents
- Abstract = i
List of tables = vi
List of figures and schemes = vii
List of Abbreviations = ix
Introduction = 1
1. History and classification of hypersensitivities = 1
2. Components of allergic reactions = 5
2.1. Allergen = 5
2.2. Allergic antibody, IgE = 8
2.3. High affinity receptor for IgE (FcεRI) = 9
2.4. Role of FcεRI in allergic disorders = 12
2.5. Low affinity receptor for IgE (FcεRII) = 12
2.6. Mast cells = 13
2.7. Basophils = 13
2.8. Intracellular events in mast cells and basophils degranulation = 15
3. Agents mediated type I hypersensitivity = 19
3.1. Histamine = 19
3.2. Leukotrienes and prostaglandins = 20
3.3. Cytokines = 20
3.4. Type I hypersensitivity can be systemic or localized = 20
4. Therapeutic methods for allergic reactions = 21
Materials and methods = 25
1. Materials = 25
2. Methods = 25
2.1 Culture of animal cells = 25
2.2 Preparation of marine algae extract = 25
2.3 Extraction and purification of phlorotannins = 26
2.4 Spectroscopic measurements = 26
2.5 Sample preparation for cell treatment = 26
2.6 Cytotoxic assessment using MTT assay = 27
2.7 Histamine release assay on KU812F and RBL-2H3 cell using = 27
2.8 Histamine release assay on KU812F cell mediated by human = 27
2.9 Histamine measurement = 27
2.10 β-hexosaminidase release assay on RBL-2H3 mediated by = 28
2.11 Total RNA extraction from KU812F cell = 29
2.12 Reverse-transcription polymerase chain reaction (RT-PCR) = 29
2.13 Western blot analysis of protein expression = 29
2.14 Flow cytometry analysis of binding activity between human = 30
2.15 Flow cytometry analysis of FcεRI α subunit expression on = 30
2.16 Flow cytometry analysis of histamine binding activity on = 30
2.17 Statistical analysis = 31
Results and Discussions = 33
1. Screening results of anti-allergic effects of marine algae = 33
1.1 Inhibition of histamine release from methanol extract of marine = 33
1.2 Inhibition of histamine release from E. cava fractions on KU812F = 33
1.3 Effect of E. cava extract on surface expression of FcεRI receptors = 37
1.4 Effect of E. cava methanolic extract on FcεRI α chain mRNA = 37
1.5 Effects of E. cava extract on binding between IgE and cell = 37
1.6 Effect of E. cava methanolic extract on IgE mediated histamine = 37
2. Isolation, purification and elucidation structure of phlorotannins from = 43
2.1 Isolation and purification of phlorotannins from Ethyl acetate = 43
2.2 Elucidation chemical structures of phlorotannins = 43
3. Effect of phlorotannins on the degranulation of basophils = 53
3.1 Cytotoxic effects of phlorotannins on human basophilic = 53
3.2 Inhibition of histamine release on KU812F cell mediated by = 53
3.3 Inhibition of histamine release on KU812F cell mediated by = 53
3.3 Inhibition of histamine release on RBL-2H3 cell mediated by = 53
3.4 Inhibition of β-hexosaminidase release on RBL-2H3 cell mediated = 54
3.4 Inhibition on binding activity between human IgE and FcεRI = 54
4. Effect of phlorotannins expression level of FcεRI α chain on KU812F = 67
4.1 Effect of phlorotannins on cell surface expression of FcεRI using = 67
4.2 Effect of phlorotannins on protein expression level of FcεRI α chain = 67
4.3 Effect of phlorotannins on gene expression level of FcεRI α chain = 68
4.4 Effect of phlorotannins on FcεRI-mediated histamine release = 68
5. Anti-histamine effect of phlorotannins on U937 and HL-60 cells = 75
5.1 Cytotoxic effects of phlorotannins on human monocytic cell = 75
5.2 Inhibition on histamine binding activity to its receptors on U937 = 75
5.3 Inhibition on histamine binding activity to its receptors on HL-60 = 76
Summary = 84
References = 85
Acknowledgements = 99
- Degree
- Master
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