B16F10 흑색종 세포 및 RAW 264.7 대식세포에서 야생콩잎 (Glycine soja CWS1140) 추출물의 멜라닌 생성 억제 및 항염증 효과 = Anti-melanogenic and anti-inflammatory effects of wild bean (Glycine soja CWS1140) leaf extract in B16F10 melanoma cells and RAW 264.7 macrophage cells

국문 초록 (Abstract)

B16F10 흑색종 세포 및 RAW 264.7 대식세포에서 야생콩잎 (Glycine soja CWS1140) 추출물의 멜라닌 생성 억제 및 항염증 효과 이유정 생명공학과 천연물화학전공 선문대학교 일반대학원 야생콩잎(Glycine soja CWS1140)은 야생콩의 부산물로 연구가 부족했 기 때문에 본 연구에서는 B16F10 흑색종 세포와 RAW 264.7 대식세포를 이용하여 야생콩잎 추출물(CWS1140)의 멜라닌 생성 억제 및 항염증 효과 를 평가하였다. CWS1140은 DPPH와 ABTS 라디칼 소거 활성 분석을 통 해 우수한 항산화 활성을 가지고 있으며, RAW 264.7 세포에서 염증 매개 물질의 분비를 억제하는 효과를 확인하였다. 세포 독성이 나타나지 않는 농 도에서 CWS1140은 농도 의존적으로 NO, PGE2, 전염증성 사이토카인의 분 비를 억제하고, iNOS와 COX-2의 mRNA 발현을 감소시켰다. 이러한 효과 는 NO와 PGE2의 생성 감소 및 iNOS와 COX-2 단백질 발현 억제를 통해 이루어졌다. 멜라닌 합성과 관련된 연구에서는 CWS1140이 B16F10 흑색종 세포에서 멜라닌 생성을 억제하며, MITF 발현 감소를 유도하여 TRP-1, TRP-2 및 티로시나아제의 발현을 조절함을 확인하였다. 특히, CWS1140은 MAPK 신호전달 경로 (mitogen activated protein kinase)의 활성을 억제하 고, protein kinase B의 인산화를 감소시켜 멜라닌 생성을 억제하는 기전을 보였다. 이러한 결과를 통해 CWS1140은 멜라닌 합성 억제를 유도하는 미 백 관련 기능성 소재로서의 가능성을 제시한다.

다국어 초록 (Multilingual Abstract)

The tyrosinase inhibitory, anti-inflammatory, and antioxidant effects of wild bean (Glycine soja CWS1140) leaf extract on B16F10 melanoma cells and RAW 264.7 macrophage cells were evaluated. G. soja leaves are a by-product of G. soja; thus, there is nao prior research on this topic. Strong DPPH and ABTS radical-scavenging activities were observed for CWS1140. After inducing melanin production with α-MSH in B16F10 melanoma cells, treatment with CWS1140 at concentrations of 25, 50, and 100 µg/mL demonstrated concentration-dependent inhibition of melanin production and tyrosinase expression, indicating inhibition of the expression of melanogenesis-related proteins. Evaluation of the anti-inflammatory activity of CWS1140 using RAW 264.7 macrophage cells revealed concentration-dependent inhibition of NO production. NO production was inhibited through the inhibition of iNOS expression, a protein responsible for NO synthesis. These results confirmed the whitening and anti-inflammatory activities of CWS1140, suggesting its potential as a functional cosmetic material. Keyword : B16F10 melanoma cell, CWS1140, MAPK pathway, RAW 264.7 macrophage cell, wild Glycine soja leaf

목차 (Table of Contents)

• Ⅰ. Introduction
• Ⅱ. Materials and Methods
• 1. Manufacture of CWS1140 wild Glycine soja leaf extract
• 2. Materials and cell cultures
• 3. Measurement of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
• 4. Measurement of ABTS radical scavenging activity
• 5. Cytotoxicity measurement
• 6. Measurement of nitric oxide (NO) production inhibitory activity
• 7. Measurement of PGE2 production inhibitory activity
• 8. iNOS, COX-2 analysis
• 9. Measurement of inhibitory activity against the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β)
• 10. Measurement of melanin contents
• 11. Measurement of tyrosinase activity
• 12. Western blot analysis
• 13. Human skin patch test
• 14. Statistical processing
• Ⅲ. Results
• 1. Full screening of wild Glycine soja leaf extracts
• 2. Effect of CWS1140 on DPPH radical scavenging activity
• 3. Effect of CWS1140 on ABTS radical scavenging activity
• 4. Cytotoxicity evaluation
• 5. Effect of CWS1140 on nitric oxide (NO) production inhibitory activity
• 6. Effect of CWS1140 on prostaglandin E2 (PGE2) synthesis inhibitory activity
• 7. Effect of CWS1140 on iNOS and COX-2 expression inhibition using western blot
• 8. Measurement of inhibitory activity against the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β)
• 9. Effect of CWS1140 on melanin production inhibitory activity
• 10. Effect of CWS1140 on tyrosinase production inhibitory activity
• 11. Effect of CWS1140 on Protein Expression of MITF and Melanogenic Enzymes
• 12. CWS1140 suppresses p-ATK, p-GSK-3β and increases p-JNK, p-p38 and p-ERK exptrssion in the MAPK Pathway
• 13. Safety of CWS1140 on human skin
• Ⅳ. Discussion
• Ⅴ. Reference
• Ⅵ. Abstract in English