Anti-inflammatory effect of isoegomaketone isolated from radiation mutant Perilla frutescens var.crispa

학위논문 (박사)-- 서울대학교 대학원 생활과학대학 식품영양학과, 2017. 8. 한성림.About 165 lines of radiation-induced mutant P. frutescens var. crispa were screened for their anti-inflammatory activities. Among those screened, the one mutant with the highest inhibitory activity on NO production in lipopolysaccharide (LPS)-treated RAW264.7 cells was selected. The enhanced anti-inflammatory activity of the mutant seemed to be due to the increase in isoegomaketone (IK) content. IK has been shown to exhibit several biological activities including anti-inflammatory, anti-cancer, and anti-obesity effects. The induction of heme oxygenase-1 (HO-1) seemed to contribute to anti-inflammatory activity of IK in RAW264.7 cells. However, there were no studies which investigated the mechanism of HO-1 induction by IK. In the present study, anti-inflammatory effect of IK isolated from radiation mutant P. frutescens var. crispa (Antisperill) was investigated using in vitro and in vivo models. In addition, in an effort to investigate any potentiality of radiation mutant P. frutescens var. crispa as functional food, optimal extraction method was chosen, as well as anti-arthritic properties of the extracts was investigated in CAIA model. In Study 1, the mechanism of IK-induced HO-1 expression was investigated using RAW264.7 cells. RAW264.7 cells were treated with IK (5, 10, 15 M) to study the mechanism of IK-induced HO-1 expression. IK upregulated HO-1 mRNA and protein expression in a dose dependent manner. IK-induced HO-1 mRNA expression was suppressed only by SB203580, a specific inhibitor of p38 MAPK. ROS scavengers (N-acetyl-L-cysteine, NAC, and glutathione, GSH) also blocked the IK-induced ROS production and HO-1 expression. Both NAC and SB203580 suppressed the IK-induced Nrf2 activation. In Study 2, whether IK has an anti-arthritic activity in collagen antibody-induced arthritis (CAIA) animal model was investigated. Rheumatoid arthritis was induced in male Balb/c mice by collagen-antibody injection. Experimental animals were randomly divided into five groups: normal, CAIA, CAIA + IK (5 mg/kg/day), CAIA + IK (10 mg/kg/day), and CAIA + apigenin (16 mg/kg/day) and respective treatments were administered via oral gavage once per day for 4 days. Mice treated with IK (10 mg/kg/day) showed improvement in disease outcome. Arthritic score, paw volume, and paw thickness were significantly lower compared to the control CAIA mice at day 7 (73%, 15%, and 14% lower, respectively). Furthermore, histopathological examination of ankle for inflammation showed that infiltration of inflammatory cells and edema formation were reduced by IK treatment. Similarly, neutrophil to lymphocyte ratio (NLR) in whole blood was lower in mice treated with IK (10 mg/kg/day) by 51.9% compared to the control CAIA mice. In Study 3, to determine the optimal extraction method for developing radiation mutant P. frutescens var. crispa as functional food, extracts were obtained by two methods: extract obtained by supercritical carbon dioxide extraction (SFE) and extract obtained by ethanol extraction (EE). SFE contained 5-fold higher levels of IK compared with EE. When LPS-induced RAW264.7 cells were treated with extracts at 25 g/mL, the SFE inhibited the expression of inflammatory mediators such as nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), interleutkin-6 (IL-6), interferon- (IFN-), and inducible nitric oxide synthase (iNOS) to a much greater extent compared with EE. In Study 4, whether SFE (in Study 3) has an anti-arthritic activity in collagen antibody-induced arthritis (CAIA) animal model was investigated. Extracts were obtained by supercritical carbon dioxide extraction method from radiation mutant P. frutescens var. crispa leaf (SFE-M) and from wild type species leaf (SFE-W). Experimental animals were randomly divided into four groups: normal, CAIA, CAIA + SFE-M (100 mg/kg/day), and CAIA + SFE-W (100 mg/kg/day) and respective treatments were administered via oral gavage once per day for 4 days. Mice treated with SFE-M showed improvement in disease outcome. Arthritic score, paw volume, and paw thickness were significantly lower compared to the control CAIA mice from day 3 to day 7. Furthermore, histopathological examination of ankle for inflammation showed that infiltration of inflammatory cells and edema formation were reduced by SFE-M treatment. Similarly, NLR in whole blood was lower in mice treated with SFE-M by 37% compared to the control CAIA mice. However, SFE-W didnt show any significant effect compared to the control CAIA group. IK showed anti-inflammatory properties by HO-1 expression via ROS/p38 MAPK/Nrf2 pathway in RAW264.7 cells, as well as real, actual, and palpable anti-arthritic effect in CAIA animal model. Furthermore, supercritical carbon dioxide extraction was found to be the better method compared with the ethanol extraction method for the presentation of extract from leaves of radiation mutant P. frutescens var. crispa to be used as functional food because of higher IK content. Efficacy of the extract from radiation mutant P. frutescens var. crispa by SFE was confirmed as the treatment with the extract reduced the incidence of clinically evident signs and symptoms in CAIA animal model. Taken together, the results of this study encourage the commercial use of the extract of radiation mutant P. frutescens var. crispa as a functional food in the chronic inflammatory situation like RA.I. Introduction 1 1. Background 2 2. Objectives of the study 5 II. Literature review 7 1. Radiation breeding 8 2. Characteristics of Perilla frutescens 13 2.1. Bioactivity 13 2.2. Active constituents 16 2.3. Extraction method 18 3. Pharmacological activities of isoegomaketone 20 4. Mouse model of rheumatoid arthritis 21 4.1. Collagen-induced arthritis (CIA) 22 4.2. Collagen antibody-induced arthritis (CAIA) 23 4.3. Zymosan-induced arthritis 23 5. Previous study 24 III. Study 1: Isoegomaketone upregultes heme oxygenase-1 in RAW264.7 cells via ROS/p38 MAPK/Nrf2 pathway 28 1. Abstract 29 2. Introduction 30 3. Materials and Methods 32 3.1. Reagents 32 3.2. Isolation of IK 32 3.3. Cell culture 38 3.4. Cytotoxicity assay 38 3.5. Determination of NO concentration 38 3.6. Preparation of cell extracts and western analysis 39 3.7. Quantitative real-time PCR 40 3.8. Statistical analysis 43 4. Results 44 4.1. Effect of IK on HO-1 expression in RAW264.7 cell 44 4.2. Effect of kinase inhibitors on HO-1 expression by IK treatment 46 4.3. Effect of ROS scavengers on HO-1 expression by IK treatment 49 4.4. Effect of ROS scavenger and p38 MAPK inhibitor on Nrf2 activation by IK treatment 51 4.5. Effect of NAC on NO production in LPS-treated RAW264.7 cells 53 4.6. Effect of ROS scavengers on the production of antioxidant enzymes 55 5. Discussion 57 IV. Study 2: Isoegomaketone alleviates the development of collagen antibody-induced arthritis in male Balb/c mice 61 1. Abstract 62 2. Introduction 63 3. Materials and Methods 65 3.1. Animals 65 3.2. Sample preparation 65 3.3. Collagen antibody-induced arthritis 65 3.4. Assessment of clinical signs of inflammation 68 3.5. Histopathological assessment 68 3.6. Analysis of neutrophil and lymphocyte 69 3.7. Statistical analysis 69 4. Results 70 4.1. Effect of IK treatment on development of RA in CAIA model 70 4.2. Effect of IK treatment on paw volume in CAIA model 74 4.3. Effect of IK treatment on paw thickness in CAIA model 76 4.4. Effect of IK treatment on arthritic score in CAIA model 78 4.5. Effect of IK treatment on blood cell population in CAIA model 80 5. Discussion 82 V. Study 3: Comparison of the anti-inflammatory activities of supercritical carbon dioxide versus ethanol extracts from leaves of radiation mutant Perilla frutescens var. crispa 86 1. Abstract 87 2. Introduction 88 3. Materials and Methods 90 3.1. Materials 90 3.2. Cell culture 90 3.3. Ethanol extraction 91 3.4. SC-CO2 extraction 91 3.5. HPLC analysis 92 3.6. Cytotoxicity assay 92 3.7. Determination of NO concentration 93 3.8. Preparation of cell extracts and western blot analysis 93 3.9. Quantitative real-time PCR 95 3.10. Measurement of MCP-1, IFN-, and IL-6 by ELISA 97 3.11. Statistical analysis 97 4. Results 98 4.1. Yield and composition of SFE (supercritical carbon dioxide extract) and EE (ethanol extract) 98 4.2. Effect of SFE and EE on cell viability 102 4.3. Effect of SFE and EE on LPS-stimulated NO production in RAW264.7 cells 104 4.4. Effect of SFE and EE on production of inflammatory mediators in LPS-stimulated RAW264.7 cells 107 5. Discussion 110 VI. Study 4: Anti-arthritic activities of the supercritical carbon dioxide extract from radiation mutant Perilla frutescens var. crispa in collagen antibody-induced arthritis 113 1. Abstract 114 2. Introduction 115 3. Materials and Methods 118 3.1. Animals 118 3.2. SC-CO2 extraction 118 3.3. HPLC analysis 119 3.4. Sample preparation and treatment 119 3.5. Collagen antibody-induced arthritis 120 3.6. Assessment of clinical signs of inflammation 122 3.7. Histopathological assessment 122 3.8. Analysis of neutrophil and lymphocyte 123 3.9. Statistical analysis 123 4. Results 124 4.1. Composition of SFE-M (supercritical carbon dioxide extract from radiation mutant P. frutescens) and SFE-W (supercritical carbon extract from wild type P. frutescens) 124 4.2. Effect of SFE-W and SFE-M treatment on development of RA in CAIA model 126 4.3. Effect of SFE-W and SFE-M treatment on paw volume in CAIA model 131 4.4. Effect of SFE-W and SFE-M treatment on paw thickness in CAIA model 133 4.5. Effect of SFE-M and SFE-W treatment on arthritic score in CAIA model 135 4.6. Effect of SFE-M and SFE-W treatment on blood cell population in CAIA model 137 5. Discussion 139 VII. Overall Discussion 142 References 147 국문초록 163Docto

Streptomyces exfoliatus SMF19에서 생산되는 B-lactamase inhibitor의 특성 및 생리적 역할

학위논문(석사)--서울대학교 대학원 :생명과학부,2001.Maste