헴프(Cannabis sativa L.)의 일본뇌염 바이러스에 대한 항바이러스활성 및 기작 분석 = Extracts and Organic Solvent Fractions from Cannabis sativa L. (Hemp) Stems and Roots against Japanese Encephalitis Virus

다국어 초록 (Multilingual Abstract)

Japanese encephalitis virus (JEV) poses significant public health challenges due to the lack of therapeutic options. With a view to developing new antiviral agents against JEV, we evaluated antiviral activity of ethanol extracts and organic solvent fractions prepared from hemp (Cannabis sativa L.). Ethanol fractions were prepared from the roots and stems of hemp and organic solvent fractions were prepared from the ethanol extracts. Noncytotoxic concentrations of the extracts and fractions were determined by in vitro cytotoxicity test. At noncytotoxic concentrations, hemp extracts and fractions demonstrated potent virucidal activity against JEV, among which hexane fractions exhibited the strongest activity. Viral growth kinetics experiments indicated that hexane and chloroform fractions inhibited JEV growth when post-treated to virus-infected cells. However, the fractions did not demonstrate inhibitory activity when co-treated with the virus to cells or pre-treated to cells before viral infection. When post-treated to virus-infected cells, hexane and chloroform fractions suppressed JEV mRNA expression in the cells. In addition, Western blot confirmed the inhibition of JEV E protein expression by post-treatment of chloroform fraction. GC-MS analysis confirmed that hemp fractions contained various compounds, 15 of which matched those previously reported to be present in hemp. Among hemp-derived compounds, stigmasterol was shown to have potent virucidal activity against JEV, suggesting that stigmasterol is one of virucidal agents in the hemp. Our study suggests that, with potent virucidal activity and antiviral activity against JEV, hemp-derived natural products provide an attractive resource for developing antiviral or therapeutic options against JEV.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 5
• 1. 실험재료 5
• 1.1. 헴프 줄기 및 뿌리로부터의 에탄올 추출 및 유기용매 분획물 조제 ··5
• 1.2. 세포주, 일본뇌염 바이러스, 천연물 7
• 2. 실험방법 7
• 2.1. 세포독성 평가 7
• 2.2. 일본뇌염 바이러스 plaque assay 8
• 2.3. 바이러스사멸활성 9
• 2.4. 일본뇌염 바이러스 성장 동역학 분석 9
• 2.5. 바이러스 부착 억제실험 10
• 2.6. Western blot 11
• 2.7. RNA 추출 및 quantitative real-time PCR 12
• 2.8. 형광 면역염색 분석 14
• 2.9. In silico 분자 도킹 14
• 2.10. GC-MS 물성 분석 15
• 2.11. 통계 분석 15
• Ⅲ. 결과16
• 1. 헴프 추출물 및 분획물의 세포독성 분석 16
• 2. 헴프 추출물 및 분획물의 일본뇌염 바이러스 사멸활성 18
• 3. 헴프 유기용매 분획물의 일본뇌염 바이러스 성장 억제 21
• 4. 헴프 유기용매 분획물의 일본뇌염 바이러스 mRNA 및 E 단백질 발현
• 억제 23
• 5. 헴프 분획물의 GC–MS 화학 조성 분석 26
• 6. Stigmasterol의 일본뇌염 바이러스 사멸활성 33
• 7. Stigamsterol의 일본뇌염 바이러스 세포부착 억제 36
• 8. 일본뇌염 바이러스 감염 후 stigmasterol의 바이러스 증식 억제 38
• 9. Stigmasterol과 일본뇌염 바이러스 단백질의 분자도킹 분석42
• Ⅳ. 결론 및 고찰 45
• Ⅴ. 참고문헌 49
• Ⅵ. 영문초록 56