Anti-human Papillomavirus (HPV) and Anticancer Activities of the Compounds Isolated from Eisenia bicyclis

다국어 초록 (Multilingual Abstract)

Eisenia bicyclis (Kjellman) Setchell is an edible perennial brown alga and belongs to the family Lessoniaceae in the order Laminariales. E. bicyclis is distributed along the coasts of Japan and Korea. Previous studies on E. bicyclis have investigated several biological activities including anti-inflammation, antioxidant, anti-diabetic and neuroprotective effects. We have found that the EtOH extract of E. bicyclis has anti-viral activity against human papillomavirus (HPV) and anti-cancer activity against HeLa human cervical cancer cell lines. E. bicyclis was extracted with EtOH. The EtOH extract was suspended in distilled water, and then consecutively partitioned with CH2Cl2, EtOAc and n-BuOH to give CH2Cl2, EtOAc, n-BuOH and H2O fractions. The anti-viral activity of the extract and solvent fractions was evaluated by using bioluminescence (SEAP) assay on HPV16 PVs infected 293TT cells. Among these fractions, the CH2Cl2 and EtOAc fractions showed comparatively higher activity than other fractions. Anticancer activity of the extract and solvent fractions was evaluated by using MTT assay against HeLa cell. The EtOAc fraction showed significantly higher activity than other fractions. The CH2Cl2 and EtOAc fractions were subjected to column chromatographic separation so that we isolated nine (1~9) including a new phlorotannin (3) and four (10~13) compounds from the EtOAc and CH2¬Cl2 fractions, respectively. Their structure were determined as phloroglucinol (1), eckol (2), 8,8’-biekcol (4), eckostolonol (7), phlorofucofuroeckol B (8), phlorofucofuroeckol A (9), ecklonialactone B (10), palmitic acid (11) and fucosterol (13) on the basis of spectroscopic analysis. The structure of a new phlorotannin, named bicyclol A, was proposed as 4,9-bis(3,5-dihydroxyphenoxy)dibenzo[b,e][1,4]dioxine-1,3,6,8-teraol (3) by spectroscopic analysis. Among the isolated compounds, compounds 1, 6 and 9 exhibited 63~73% reduction of HPV16 PVs at concentration 50 μg/ml. Compounds 2 and 6 showed moderate cytotoxicity with IC50 values 84.91 and 145 μg/ml, respectively. Compounds 12 and 13 isolated from the CH2Cl2 fraction, revealed cytotoxicity with IC50 values of 162.30 and 154.54 μg/ml against HeLa cell, respectively. Compound 6 showed significant anti-HPV viral and anticancer activities simultaneously.

목차 (Table of Contents)

• I. INTRODUCTION １
• 1. The Origin of Eisenia bicycles (Kjellman) Setchell １
• 2. History of research on Eisenia bicyclis 4
• 3. Human papillomavirus and cervical cancer 11
• 4. Aim of study 17
• II. MATERIALS 18
• 1. Materials 18
• 1.1 Plant material 18
• 1.2 Regents and instruments 18
• 1.2.1 Regents 18
• 1.2.2 Instrument 19
• III. METHODS 21
• 1. Extraction and Fractionation 21
• 2. Isolation of compounds from the EtOAc fraction 23
• 2.1 Isolation of compounds 1 and 2 24
• 2.2 Isolation of compounds 3, 4, 5 and 6 25
• 2.3 Isolation of compounds 7, 8 and 9 27
• 3. Isolation of compounds from the CH2Cl2 fraction 30
• 3.1 Isolation of compounds 10, 11, 12 and 13 32
• 4. Anti-viral activity 35
• 4.1 Cells 35
• 4.2 Generation of HPV16 pseudoviruses (PVs) 35
• 5. Anticancer activity 37
• 5.1 Cell lines and culture conditions 37
• 5.2 MTT assay for cell viability 37
• IV. RESULT AND DISCUSSION 39
• 1. Structures of compounds isolated from the EtOAc fraction 39
• 2. Structures of compounds isolated from the CH2Cl2 fraction 41
• 3. Anti-viral activity 43
• 3.1 Anti-viral activity of the extract and solvent fractions from E. bicyclis 43
• 3.2 Anti- viral activity of the isolated compounds 45
• 4. Anticancer activity 47
• 4.1 Cytotoxicity of the EtOH extract and solvent fractions from E. bicyclis 47
• 4.2 Cytotoxicity of the isolated compounds from the EtOAc fraction 52
• 4.3 Cytotoxicity of the isolated compounds from the CH2Cl2 fraction 53
• V. CONCLUSION 54
• VI. REFRENCE 56
• Appendix 62
• KOREAN ABSTRACT 101