섬기린초에서 분리한 항산화 및 항염증 물질 : Antioxidant and Antiinflammatory compounds from Sedum takesimense Nakai

다국어 초록 (Multilingual Abstract)

Sedum takesimense Nakai is an endemic species in Korea, and there is no report on its constituents and biological activities. One of related plants, Sedum kamtshaticum has been used as a folk medicine for anti-inflammation, and improving the circulation of blood. In this study, the methanol extract and solvent fractions of S. takesimense were ----------------------------------------------------------------
* A thesis submitted to the committee of Graduate School, Chungnam National University in a partial fulfillment of the requirements for the degree of doctor of Philosophy conferred in August 2006.
measured on several radical scavenging activities and inhibitory effect of COX-1 and COX-2. Among them, ethyl acetate and butanol fractions which showed strong anti-oxidant activity and relatively effective inhibitory COX-1 and COX-2 effects were selected to isolate the bio-active constituents.
In order to isolate bio-active constituents, fresh whole plant of S. takesimense (2.5 kg) was refluxed with MeOH for 4 h. The MeOH extract (460g) was suspended in water and then partitioned with hexane, ethyl acetate, and butanol, sequentially. The IC50 (μg/ml) values of DPPH and superoxide radical scavenging activity of ethyl acetate fraction were 4.6 and 0.7, and the IC50 (μg/ml) of butanol fraction were 5.2 and 0.7, respectively. Additionally, COX-1 and COX-2 inhibitory effects of ethyl acetate fraction were 84.7% and 91.6% at 200 μg/ml as the final concentration. Furthermore, COX-1 and COX-2 inhibitory effects of butanol extract were 68.8% and 75.7%. Therefore, these two fractions selected to isolate the bio-active constituents.
Sephadex gel and silca gel column chromatography, and preparative HPLC of the active fractions led to the isolation of fourteen compounds including three new compounds. They were identified as ferulic acid (1), caffeic acid (2), gallic acid (3), methyl gallate (4), 1-(4-hydroxyphenyl) - 2-(3,5-dihydroxyphenyl)2-hydroxyethanone (5), myricetin (6), quercetin (7), luteolin (8), rhodalin (9), gossypetin-8-O-β-D-xyloside (10), rhodalidin (11), luteolin-7-O-β-glucoside (12), 2,6-di-O-galloylarbutin (13), and arbutin (14) by comparing spectral data with those previously reported. Among them, compound 5, 10, and 13 were isolated as new compounds.
The isolated compounds were tested for their anti-oxidative activities with DPPH and superoxide radical scavenging assay system. 2,6-di-O-galloylarbutin (13) showed strongest DPPH radical scavenging activity and the IC50 value was 3.63 μM. Gossypetin-8-O-β-D-xyloside (10) also showed prominent superoxide radical scavenging activity and the IC50 value was 5.5 μM. These two new compounds have effective scavenging activity on each other free radical. In the NO radical scavenging assay, well known compounds such as caffeic acid and luteolin showed relatively effective NO radical scavenging activity. On the basis of these antioxidant activity, three new compounds from S. takesimense were evaluated for inhibitory effects of LDL-oxidation. As results, IC50 values of 10 and 13 in TBARS assay were 5.7 and 3.3 μM, respectively. In addition, the lag time (10: 72 and 180 min 13: 80 and 170 min, probucol: 76 and 120 min at 1 and 3 μM, and control: 41 min) and the REM (inhibition of 10 and 13: 100% and 38% at 10 μM). In conclusion, the antioxidant activity of 2,6-di-galloylarbutin (13) showed more potent than positive controls in these assay.
Two new compounds, 10 and 13 showed strong inhibitory effects on COX-1 activity with the IC50 values of 32.2 and 55.7 μM, respectively. Meanwhile, compound 5 exhibited supressive activity against both COX-1 and COX-2 activities, with the IC50 values of 39.8 and 61.4 μM. From these, the anti-inflammatory action of three new compounds might be related to the inhibitory effect of cyclooxygenase. In addition, the inhibitory effect of LPS-induced NO and ROS production in RAW 254. 7 cell and cell viability for 5, 9, 10, and 13 were evaluated. All compounds show no cytotoxicity to RAW 264.7 cells. Compound 9, 10 and 13 showed inhibitory effect of LPS-induced ROS production; the percent inhibitions of ROS production were 19, 39 and 71% for 10, 39, 53 and 69 % for 13, 66, 66 and 72 % for 9 at 50, 100, and 200 μM , respectively. Three new compounds showed inhibitory effect of TPA-induced mice ear edema and lymph node in vivo. The percent inhibition of TPA-induced mice ear edema for 5, 10, and 13 were 65.3, 48.7, and 69.7 %, respectively. Additionally, the percent inhibition of TPA-induced mice lymph node's weight for 5, 10, and 13 were 66.7, 33.3, and 33.3 %. However, it was expressed that there was no remarkable inhibition of TNF-α formation except for 13, which showed 21.7 % inhibition on TNF-α formation.

목차 (Table of Contents)

• I. INTRODUCTION 1
• 1. The plant Sedum takesimense Nakai 1
• 1.1. Other Sedum Genus 2
• 2. Oxidative damage and Pathogenesis 6
• 2.1. The Consequences of generation of ROS 6
• 2.1.1. Lipid peroxidation 6
• 2.1.2. Damage of DNA and Protein 7
• 2.1.3. ROS and Antioxidants 8
• 2.2. The role of nitric oxide 9
• 2.3. Antioxidants and atherosclerosis 10
• 2.3.1. Inhibition of LDL oxidation by antioxidants 12
• 2.3.2. Inhibition of ACAT and atherosclerosis 13
• 3. Inflammation and Pathogenesis 15
• 3.1. Cyclooxygenase and inflammation 15
• 3.2 TNF-α and inflammation 17
• 3.3. Lp-PLA2: a producer of inflammatory mediators 20
• II. Materials and Methods 22
• 1. Plant material 22
• 2. Reagents and Instruments 22
• 2.1. Reagents 22
• 2.2 Instrument 23
• 3. Methods 24
• 3.1 Extraction and Fractionation 24
• 3.2 Isolation of compounds 25
• 3.2.1 Compound 1
• 3.2.2 Compound 2
• 3.2.3 Compound 3
• 3.2.4 Compound 4
• 3.2.5 Compound 5
• 3.2.6 Compound 6
• 3.2.7 Compound 7
• 3.2.8 Compound 8
• 3.2.9 Compound 9
• 3.2.10 Compound 10
• 3.2.11 Compound 11
• 3.2.12 Compound 12
• 3.2.13 Compound 13
• 3.2.14 Compound 14
• 3.3 Acid Hydrolysis of sugar 34
• 3.4 Biological Assay 35
• 3.4.1. DPPH radical scavenging assay 35
• 3.4.2. Superoxide radical scavenging assay 35
• 3.4.3. NO radical scavenging assay 36
• 3.4.4. Inhibitory assay of lipid peroxidation 37
• 3.4.4.1 Preparation of mitochondria 37
• 3.4.4.2 Oxidation of mitochondria 37
• 3.4.5. Inhibitory assay of LDL-oxidation 38
• 3.4.5.1. Blood collection 38
• 3.4.5.2 Cu2+induced oxidation of LDL 39
• 3.4.5.3. Measurement of conjugated dienes 39
• 3.4.5.4. Measurement of relative electrophoretic mobility(REM) 40
• 3.4.6. Inhibitory assay of ACAT 40
• 3.4.7. Inhibitory assay of COX-1 and COX-2 40
• 3.4.8. Inhibitory assay of Lp-PLA2 41
• 3.4.9. Inhibitory assay of cell - mediated NO and ROS production 42
• 3.4.9.1. Cell culture 42
• 3.4.9.2. Cell viability 43
• 3.4.9.3. Nitirite assay 43
• 3.4.9.4. Determination of ROS production 44
• 3.4.10. Acute phorbol ester(TPA)-induced inflammation assay 44
• 3.4.10.1. Animal 44
• 3.4.10.2. TPA-induced ear edema of mouse 44
• 3.4.10.3. Measurement of TNF-α 45
• Ⅲ. Results and Discussion 46
• 1. Biological properties of fractions of S. takesimense 46
• 1.1 DPPH and Superoxide radical scavenging activity 46
• 1.2 Inhibitory effect of Lipid peroxidation and COX-1 and 2 47
• 2. Determination of chemical structure 49
• 2.1. Compound 1 49
• 2.2. Compound 2 50
• 2.3. Compound 3 51
• 2.4. Compound 4 52
• 2.5. Compound 5 53
• 2.6. Compound 6 60
• 2.7. Compound 7 62
• 2.8. Compound 8 63
• 2.9. Compound 9 65
• 2.10. Compound 10 71
• 2.11. Compound 11 80
• 2.12. Compound 12 89
• 2.13. Compound 13 91
• 2.14. Compound 14 98
• 3. Biological properties of Isolated phenolic compounds 100
• 3.1 Effect of phenolic compounds on DPPH and Superoxide radical scavenging activity 100
• 3.2 Effect of phenolic compounds on NO radical scavenging activity 102
• 3.3 Effect of phenolic compounds on Cu2+-mediated LDL-oxidation 103
• 3.3.1. Effects on TBARS formation 103
• 3.3.2. Effects on conjugated on conjugated diene formation 105
• 3.3.3. Effects on relative electrophoretic mobility (REM) 106
• 3.4 Effect of phenolic compounds on ACAT 108
• 3.5 Effect of phenolic compounds on COX-1 and COX-2 109
• 3.6 Effect of phenolic compounds on Lp-PLA2 110
• 3.7 Effect of phenolic compounds on NO and ROS production in the cell 111
• 3.7.1 Effect on cytotoxicity in RAW 254. 7 cell 111
• 3.7.2. Effects on LPS-induced NO production in RAW 254. 7 cell 111
• 3.7.3. Effects on LPS-induced ROS production in RAW 254. 7 cell 113
• 3.8 Effect of phenolic compounds on acute inflammation 115
• Ⅳ. Conclusion 118
• Reference 121
• Abstract in English 134
• Abstract in Korean 138