Physiological Activities of Phthalate Derivatives Isolated from Seahorse, Hippocampus Kuda Bleeler
- Alternative Title
- 해마로부터 분리한 프탈레이트 유도체들의 생리활성
- Abstract
- 해마는 큰가시고기목 실고기과의 동물로서 내장을 제외한 전체부분을 양위, 이뇨와 항노화 등에 쓰이는 생약재로서 동부 아시아 에서 많이 사용되고 있다. 본 연구에서는 해마의 MeOH 추출물을 가지고 Silica gel column chromatography, HPLC 와RP-18 gel을 사용하여 성분을 분리하고, 분리된 화합물의 구조는 IR, UV, 1H-NMR, 13C-NMR, HMBC, HMQC 및 EIMS 등의 분광학적 방법을 이용하여 화합물 1-3을 분리. 동정하였다. 분리한 화합물은 천연물질에서 처음 분리 동정 된 2-ethyldecyl 2-ethylundecyl phthalate와 2, 12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecylphthalate 그리고 합성에서 존재하는 물질 Bis (2-ethylheptyl) phthalate로 동정되었다. 다음 분리한 화합물을 항산화성, β-secretase 저해효과, 고혈압 저해효과, Tyrosinase 저해 효과 등 생리활성에 대해 연구하였다.
항산화활성은 DPPH, 하이드록시, 수퍼옥사이드, 알킬 라디칼 및 DCF-DA을 이용하여 측정한 결과 화합물 2 이 우수한 소거능을 나타냈었고, 알즈하이머성 치매 질활 유발 인자인 β-secretase 저해효과를 측정한 결과 일정한 저해활성이 나타났었고 그들 중 화합물 2이 저해활성이 가장 우수함을 확인하였다. 또한 고혈압 저해효과와 Tyrosinase 저해 양상을 조사한 결과 모두 경쟁적 저해를 하는 것으로 나타났으며 화합물 2가 가장 우수한 저해활성을 지닌 것으로 나타났다. 그리고 여러가지 세포를 이용한 독성실험에서도 세포독성을 지니지 않은 것으로 나타내었다.
Seahorse, Hippocampus kuda a marine teleost fish, is well known not only for its special medicinal composition and but also as one of the most famous and expensive materials of traditional Chinese medicine. H. Kuda Bleeler was extracted with methanol at room temperature to give the methanol extract. This extract was subjected to SiO₂ gel flash chromatography, followed by C-18 ODS gradient chromatography and reverse-phase HPLC, to yield three phthalate derivatives, two new compounds (1, 2) and one natural known compound (3). The three derivatives are 2-ethyldecyl 2-ethylundecyl phthalate (1), 2,12-diethyl-11-methyl
hexadecyl 2-ethyl-11-methylhexadecyl phthalate (2) and bis(2-ethylheptyl) phthalate (3). The structures of these compounds were elucidated mainly by the comprehensive analysis of their NMR spectroscopic data.
Antioxidant activities of derivatives were investigated for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl, superoxide, peroxyl radical, and hydrogen peroxide using electron spin resonance (ESR) spectroscopy. Among the compounds, 2 exhibited the highest antioxidant activity in linoleic acid system, DPPH radical scavenging, hydroxyl radical scavenging, superoxide radical scavenging, peroxyl radical scavenging, and inhibitory intracellular ROS. Furthermore, MTT assay showed no cytotoxicity on mouse macrophages cell (RAW264.7) and human fetal lung fibroblast cell line (MRC-5). The tested beta-scretase inhibitoty activity, compound (1-3) inhibition, and the IC50 values were 0.722, 0.392, and 1.12 mM, respectively, and the inhibitory pattern were found to non-competitive. Among the compounds, 2 showed the strongest angiotensin I converting enzyme (ACE) inhibitory activity, and the inhibitory pattern was found to be competitive. The IC_(50) values of compound 1, compound 2 and compound 3 were 63.9 μM, 33.6 μM and 153.6 μM, respectively. The tyrosinase inhibitory activity of compounds, final concentrations of 0.0225, 0.1125, 0.225 and 0.45 of compounds were tested for enzyme inhibitory activity and it was observed that it could inhibit tyrosinase at IC50 of 0.624, 0.382, and 1.15 mM, respectively. The results showed that compound 2 could exhibit tyrosinase activity by approximately 63.2% higher than those of compound 1 and 3. The inhibitory pattern was found to be non-competitive. Moreover, compounds inhibited production of major inflammatory mediators such as nitric oxide (NO) and prostaglandin E₂ (PGE₂) and the expression of corresponding enzymes, including iNOS and COX-2 in RAW264.7 mouse macrophage cells exposed to bacterial lipopolysaccharide (LPS).
- Issued Date
- 2008
- Awarded Date
- 2008. 2
- Type
- Dissertation
- Publisher
- 부경대학교 대학원
- Alternative Author(s)
- Qian, Zhong-Ji
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract = i
Table of Contents = iii
List of Charts = ix
List of Schemes = ix
List of Figures = x
List of Tables = xv
List of Abbreviations = xvii
Chapter1 Extraction and Isolation of compound derivatives from Seahorse = 1
1. Introduction = 2
2. Experimental Procedures = 5
2.1. Chemical reagent = 5
2.2. Isolation and extraction of the seahorse = 6
2.3. Thin-Layer Chromatography (TLC) = 8
2.4. Infrared Spectrum = 8
2.5. Ultraviolet and Visible (UV-Vis) Spectrophotometry = 8
2.6. Mass spectrometry = 8
2.7. Specific rotation?[α]D = 8
2.8. Nuclear Magnetic Resonance (NMR) = 9
3. Results and Discussions = 9
3.1. Structure of Compounds = 9
3.2. Structural elucidation of the compounds = 13
3.2.1. Compound (1) = 13
3.2.2. Compound (2) = 20
3.2.3. Compound (3) = 29
4. References = 36
Chapter2 Physiological Activities of compounds = 40
1. General Introduction = 41
1.1. Antioxidant Effects = 41
1.2. Beta-secretase inhibition effects = 44
1.3. ACE inhibition effects = 45
1.4. Tyrosinase inhibition effects = 46
1.5. Anti-inflammatory effects = 48
2. Materials and Methods = 51
2.1. Materials = 51
2.2. Antioxidant activities of compounds = 52
2.2.1. Lipid peroxidation inhibition assay = 52
2.2.2. Extinction of DPPH radical = 53
2.2.3. Hydroxyl radical scavenging assay = 53
2.2.4. Superoxide radical scavenging assay = 54
2.2.5. Peroxyl radical scavenging assay = 54
2.2.6. Culture of cells and viability determination = 55
2.2.7. Determination of intracellular formation of reactive oxygen = 56
2.2.8. Myeloperoxidase activity assay = 56
2.2.9. Genomic DNA isolation and Protective effect of the comp = 57
2.3. Beta-secretase inhibitory activities of compounds = 58
2.3.1. Beta-secretase inhibitory assay = 58
2.3.2. Determination of the inhibition pattern of compounds = 58
2.4. Antihypertensive effects of compounds = 58
2.4.1. Measurement of ACE inhibitory activity = 59
2.4.2. Determination of the inhibition pattern on ACE = 59
2.5. Tyrosinase Inhibition effects of compounds = 59
2.5.1. Tyrosinase enzyme inhibitory assay = 59
2.5.2. Determination of the inhibition pattern = 60
2.5.3. Cell viability assay = 60
2.5.4. Measurement of melanin = 61
2.6. Anti-inflammatory effects of compounds = 61
2.6.1. Assessment of cellular No production = 61
2.6.2. Western blotting = 62
2.7. Statistical analysis = 62
3. Results and Discussion = 62
3.1. Antioxidant effects of compounds = 62
3.1.1. Effect of compounds (1-3) on lipid peroxidation inhibition = 63
3.1.2. Free radical scavenging activity of compounds (1-3) = 63
3.1.3. Effects of compounds on intracellular reactive oxygen = 74
3.1.4. Myeloperoxidase (MPO) inhibitory effects of compounds = 80
3.1.5. DNA oxidation inhibition potential of compounds = 82
3.1.6. Structure-antioxidant activity relationship of compounds = 86
3.2. Beta-secretase inhibitory activities of compounds = 87
3.3. Antihypertensive effects of compounds = 93
3.3.2. ACE inhibitory activities of compounds = 93
3.3.2. Determination of the inhibition pattern of ACE = 95
3.4. Tyrosinase inhibitory activity of compounds = 100
3.4.1. Tyrosinase inhibitory activities and inhibition pattern = 100
3.4.2. Cell viability and melanin contents = 106
3.5. Anti-inflammatory effects of compounds = 110
Chapter3 Summary and Conclusion = 119
Chapter4 References = 123
- Degree
- Doctor
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