LPS로 유도한 염증반응과 산화 스트레스에 대한 황칠나무 에틸 아세테이스 분획의 억제 효과
Inhibitory Effect of Ethyl Acetate Fraction from Dendropanax morbifera Extracts on LPS-induced Inflammation and Oxidative Stress
- 주제(키워드) Dendropanax morbifera , Inflammation , Oxidative stress
- 발행기관 아주대학교
- 지도교수 문은표
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 석사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029911
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
In this study, the anti-inflammatory and oxidative stress inhibitory effects of Dendropanax morbifera, a native plant species in Korea, were studied. Methanol extracts of Dendropanax morbifera were fractionated according to polarity, and five fractions were named DPLS 1, DPLS 2, DPLS 3, DPLS 4 and DPLS 5 according to the solvent polarity. Five antioxidant experiments were performed on five fractions of Dendropanax morbifera and the most effective antioxidant fraction of DPLS5 was obtained. As a result, the higher polar fractions of TPC had higher polyphenol contents and the TFC showed that DPLS 1 and DPLS 2 were particularly high in flavonoids. The inhibitory effects of five fractions on the inflammation and oxidative stress were investigated using the LPS-stimulated RAW264.7 macrophages. The strong inhibitory activity against nitric oxide (NO) of DPLS 3 was observed. Therefore, pro-inflammatory cytokine secretion of DPLS 3 was analyzed and confirmed that the expression of iNOS and COX-2 was effectively reduced in the LPS-stimulated RAW264.7. In addition, DPLS 3 showed the highest inhibitory activity against intracellular ROS among the five fractions, and recovery of intracellular damage to oxidative stress was confirmed by examining mitochondrial membrane potential and antioxidant enzyme activity. To determine the mechanism of DPLS 3, the activity of inhibiting transcription factor NF-κB and MAPKs signaling pathways were further analyzed. The results confirmed that DPLS 3 inhibited the phosphorylation of IKK and IkB which suggest the repression of the NF-κB translocation from the cytoplasm to the nucleus. In addition, the inhibition of phosphorylation of p38, JNK and ERK 1/2 were confirmed, and it was expected that the transcription of AP-1 could be suppressed. In conclusion, DPLS 3, which has enhanced anti-inflammation effects and inhibition effects of oxidative stress, could be developed as a natural drug to treat diseases caused by inflammation such as rheumatoid arthritis.
more목차
Ⅰ. Introduction 1
Ⅱ. Materials and Methods 4
1. Dendropanax morbifera Extract Preparation 4
2. Phytochemical Analyses of Dendropanax morbifera Extracts 5
2.1. Measurement of Total Phenolic Compound (TPC) Content 5
2.2. Measurement of Total Flavonoid Compound (TFC) Content 5
3. Evaluation of Anti-oxidant Capacities 6
3.1. DPPH Free Radical Scavenging Assay 6
3.2. ABTS Cationic Radical Scavenging Assay 7
3.3. Hydrogen Peroxide Scavenging Assay 8
4. Cell Culture 9
5. Determination of Cell Viability 9
6. Determination of Inhibitory Effect on the inflammatory response of Dendropanax morbifera extracts 10
6.1. Measurement of Nitric Oxide Secretion 10
6.2. Measurement of IL-6 and TNF-α Secretion 10
7. Determination of Inhibitory Effects on Oxidative Stress of Dendropanax morbifera extracts 11
7.1. Detection of Intracellular ROS 11
7.2. Detection of Catalase activity 11
7.3. Detection of Mitochondrial Membrane Potential (MMP) 12
8. Western Blot Analysis 13
9. Statistical Analysis 14
Ⅲ. Results 16
1. The Yield of Extract and Fractions from Dendropanax morbifera 16
2. Phytochemical Analyses of Dendropanax morbifera Extracts 16
2.1. Total Phenolic Compound Content 16
2.2. Total Flavonoid Compound Content 17
3. Anti-oxidative Capacities of Dendropanax morbifera Extracts 20
3.1. DPPH Free Radical Scavenging Activity 20
3.2. ABTS Cationic Radical Scavenging Activity 20
3.3. Hydrogen Peroxide Scavenging Activity 21
4. Cell Viability 25
5. Inhibitory Effects on inflammatory response by LPS 27
5.1. Inhibitory Effect of Nitric Oxide secretion 27
5.2. Inhibitory Effect of IL-6 and TNF-α secretion 28
6. Inhibitory Effects on Oxidative Stress Induced by LPS 31
6.1. Inhibitory Effect of Intracellular ROS 31
6.2. Recovery Effect of Mitochondria Membrane Potential 31
6.3. Recovery Effect of Catalase activity 32
7. Inhibition of NF-κB/MAPK Signaling Pathway 36
7.1. Inhibitory Effect of iNOS and COX-2 expression 36
7.2. Inhibitory Effect of NF-κB Translocation 36
7.3. Inhibitory Effect of Phosphorylated IKK and IκB expression 37
7.4. Inhibitory Effect of phosphorylated MAPKs expression 38
Ⅳ. Discussion 44
Ⅴ. References 50
Ⅵ. 국문요약 58
