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Characterization of Phytochemicals from Cakile edentula Root Extracts that Regulate Apoptosis in Human Melanoma Cell

서양갯냉이 뿌리 추출물로부터 피부 흑색종 세포 사멸 조절 성분 규명

  • 정현정 (Hyeon-Jeong Jeong, 강릉원주대학교 일반대학원)

초록/요약 도움말

Diverse species in the genus Cakile are widely distributed across the world. In 2008, a species of Cakile plant, Cakile edentula, has been firstly found in the east coast of Korea. C. edentula is a dune plant and is also known as one of allelopathic plants. While this plant has been largely studied on their associated environments in the ecological perspective, little is known about the biological functions and chemical constituents of C. edentula. My thesis study therefore focused on the identification of chemical ingredients and the activity of bioactive compounds isolated from this plant. The root metabolome of C. edentula was extracted by ethanol, and the crude extract was subsequently solubilized by polarity-based solvent partition. The cell viability screening revealed that the methylene chloride-soluble fraction harbors potent cell cytotoxicity against a human melanoma cell line, SK-MEL-28. Employing activity-guided discovery approach, I was able to isolate the cytotoxic component. Through the high-resolution-mass spectrometry (HR-MS), Nuclear Magnetic Resonance (NMR), and the comparison with commercial standard, the chemical structure of the active component was unambiguously identified to be Iberin. I also observed that Iberin arrests G2 cycle and inhibits migration of skin melanoma cell. Additionally, I have isolated four compounds 1-4 from the root extract. By the interpretations of 2D-NMR spectra, I demonstrated all compounds to be geometrically isomerized 9-oxo-octadecatrienoic acids. 1 was a previously unreported fatty acid. While 1 did not show the cytotoxicity and apoptotic effects, 1 stimulated migration of skin melanoma cells and increased the expression levels of some pro-inflammatory biomarkers such as IL-6. This thesis study is the first report describing the presence of iberin, which has the potential to inhibit cell proliferation in a human melanoma cell line. I further demonstrated that this plant produces a series of 9-oxo-octadecatrienoic acids, one of which could be a new pro-inflammatory fatty acid.

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목차 도움말

Table of Contents
Abstract ····························································································· I
국문요약 ···························································································· III
List of Tables ······················································································ VII
List of Figures ····················································································· VIII
I. Introduction ······················································································ 1
II. Materials and Methods
1. General experimental procedures ·················································· 4
2. Plant material ············································································· 4
3. Organic extraction and solvent partition ·········································· 4
4. Isolation and purification ······························································· 5
5. Cell culture ················································································ 5
6. Cell viability ················································································ 5
7. Cell migration assay ····································································· 6
8. Annexin V-FITC staining ······························································· 6
9. Cell cycle arrest ·········································································· 6
10. Western blot·············································································· 7
11. quantitative Real-Time PCR (qRT PCR) ·········································· 7
12. Statistical analysis ······································································ 8
III. Results
1. Organic extraction and solvent partition ··········································· 9
2. Cytotoxic effects of crude extract and fractions ································· 10
3. Apoptotic effect of methylene chloride-soluble fraction ························ 11
4. First round of high-performance column chromatography and cytotoxic effects of fractions ················································································ 12
5. Second round of high-performance column chromatography and cytotoxic effects of fractions ······································································· 13
6. Third round of high-performance column chromatography and cytotoxic effects of fractions ················································································ 14
7. Fourth round of high-performance column chromatography and cytotoxic effects of fractions ······································································· 15
8. Low-resolution LS-MS and high-resolution LC-MS spectra of active compound
································································································ 16
9. Structure elucidation of active compound ·········································· 17
10. Apoptotic effect of iberin against human melanoma ···························· 18
11. Effect of iberin on melanoma cell life ················································ 19
12. Inhibitory effect of iberin on cell migration ·········································· 20
13. HPLC profile of C. edentula ···························································· 21
14. High-resolution LC-MS data of four peaks of interests ························· 22
15. Isolation and purification of four peaks of interests ······························ 23
16. Chemical structure and NMR correlations of compound 1-4 ·················· 25
17. Structure elucidation of four compounds 1 ········································· 26
18. Structure elucidation of four compounds 2 ········································· 29
19. Structure elucidation of four compounds 3 ········································· 32
20. Structure elucidation of four compounds 4 ········································· 34
21. Proposed biosynthesis of four compounds ········································ 35
22. Cell viability assay of compound 1 ··········································· 36
23. Cell cycle arrest of compound 1 ······················································ 37
24. Cell migration assay of compound 1 ················································· 38
25. Activation of compound 1 on pro-inflammatory markers ······················· 39
IV. Summary ························································································· 40
V. Reference ························································································ 41

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