臺灣博碩士論文加值系統

玫瑰草（Cymbopogon martinii）又名印度天竺葵，為禾本科植物，玫瑰草精油在香水、香料、化粧品和藥物中有廣泛的應用，主要由單萜類所組成，其主成分為香葉醇（Geraniol）和乙酸香葉酯（Geranyl acetate）。本研究利用玫瑰草精油原液和經5 %檸檬酸水進行二次蒸餾萃取獲得之改質精油為檢品，以經小鼠角質細胞（XB-2）培養之玫瑰草精油為條件培養基的方式，依照不同比例體積之條件培養基培養小鼠巨噬細胞（Raw 264.7），以進行一氧化氮含量測定來評估改質精油之抗發炎潛力，並通過酵素結合免疫吸附分析法（ELISA）評估改質精油對於促炎細胞因子含量之影響。結果顯示，經LPS誘導的Raw 264.7巨噬細胞中，玫瑰草精油原液和改質精油均可以顯著抑制一氧化氮（NO）產生，且些微降低了促炎細胞因子（TNF-α）的水平，而由條件培養基推測XB-2細胞可能會影響Raw 264.7之一氧化氮的產生，結果表明玫瑰草精油和改質精油在LPS誘導的Raw 264.7細胞中具有抗發炎潛力，且推測小鼠角質細胞分泌的細胞激素可能參與其抗發炎活性。

Cymbopogon martinii, also known as Indian geranium, belongs to the family Poaceae. Its essential oil of large applications in fragrance, perfumery, cosmetics and pharmaceuticals. The main components of Cymbopogon martinii essential oil are monoterpenes, with geraniol and geranyl acetate being the major compuunds. In this study, the modified essential oil was obtained through secondary distillation using 5% citric acid water. The modified essential oil was used to condition the culture medium for XB-2 cells, and different volumes of the conditioned medium were used to culture Raw 264.7 mouse macrophages. Nitric oxide levels were measured to evaluate the anti-inflammatory potential of the modified essential oil, and the impact of the modified essential oil on pro-inflammatory cytokine levels was assessed using enzyme-linked immunosorbent assay (ELISA). The results showed that both Cymbopogon martinii essential oil and modified essential oil could significantly inhibit the production of nitric oxide (NO) in LPS-induced Raw 264.7 macrophages. It was also slightly reduce the levels of the pro-inflammatory cytokines (TNF-α). Additionally, it is suggested from the conditioned medium that XB-2 cells may affect the production of nitric oxide in Raw 264.7 cells. The results show that both Cymbopogon martinii essential oil and modified essential oil have anti-inflammatory activity in LPS-induced Raw 264.7 cells, and it is hypothesized that cytokines secreted by mouse keratinocytes may contribute to their anti-inflammatory activity.

摘要 I
Abstract II
目錄 III
圖目錄 VII
第一章 緒論 1
一、 前言 1
二、 植物精油 2
三、 玫瑰草（Cymbopogon martinii） 3
四、 改質精油 4
五、 發炎反應 4
5-1. 角質細胞與發炎反應 5
5-2. 巨噬細胞與發炎反應 5
5-3. 自由基與發炎反應 6
5-4. 精油與抗發炎反應 8
六、 研究動機與目的 8
七、 研究架構 9
第二章 實驗材料與方法 10
一、 實驗材料 10
（一） 儀器 10
（二） 試劑套組 11
（三） 藥品 11
（四） 細胞株 13
二、 實驗方法 13
（一） 細胞培養（Cell culture） 13
1. 完全培養液配置 13
2. 冷凍細胞活化 14
3. 細胞繼代培養 14
3-1. Raw 264.7細胞繼代 14
3-2. XB-2細胞繼代 15
4. 細胞冷凍保存 16
（二） 實驗方法 17
1. 玫瑰草精油與改質精油 17
2. 條件培養基配製（Conditioned medium） 18
3. 細胞存活率測定（MTT Assay） 19
3-1. 檢品對於Raw 264.7細胞存活率測定 20
3-2. 檢品對於XB-2細胞存活率測定 20
3-3. 條件培養基對於Raw 264.7細胞存活率測定 21
4. 一氧化氮含量分析（Nitric oxide assay） 22
5. 細胞激素含量測定（ELISA kit assay） 23
5-1. 試劑配製 24
5-2. 細胞激素TNF-α含量測定 25
6. 數據分析 26
第三章 結果與討論 27
一、 細胞存活率測定 27
1-1. 精油對於Raw 264.7細胞之細胞存活率 27
1-2. 精油對於XB-2細胞之細胞存活率 31
1-3. 條件培養基對於Raw 264.7細胞之細胞存活率 34
二、 一氧化氮含量測定 37
2-1. 精油對於Raw 264.7細胞之一氧化氮含量測定 38
2-2. 條件培養基對於Raw 264.7細胞之一氧化氮含量測定 43
三、 細胞激素含量測定 63
3-1. 精油對於Raw 264.7細胞之TNF-α含量測定 64
3-2. 條件培養基對於Raw 264.7細胞之TNF-α含量測定 67
第四章 結論 72
一、 細胞存活率 72
二、 條件培養基之細胞存活率 72
三、 一氧化氮含量測定 73
四、 細胞激素含量測定 73
第五章 參考文獻 75

Ahmad, N., Ansari, M. Y., & Haqqi, T. M. (2020). Role of iNOS in osteoarthritis: Pathological and therapeutic aspects [https://doi.org/10.1002/jcp.29607]. Journal of Cellular Physiology, 235(10), 6366-6376. https://doi.org/https://doi.org/10.1002/jcp.29607
Asgari, A., & Jurasz, P. (2023). Role of Nitric Oxide in Megakaryocyte Function. Int J Mol Sci, 24(9). https://doi.org/10.3390/ijms24098145
Avoseh, O., Oyedeji, O., Rungqu, P., Nkeh-Chungag, B., & Oyedeji, A. (2015). Cymbopogon species; ethnopharmacology, phytochemistry and the pharmacological importance. Molecules, 20(5), 7438-7453. https://doi.org/10.3390/molecules20057438
Barolet, A. C., Litvinov, I. V., & Barolet, D. (2021). Light-induced nitric oxide release in the skin beyond UVA and blue light: Red & near-infrared wavelengths. Nitric Oxide, 117, 16-25. https://doi.org/10.1016/j.niox.2021.09.003
Ben Ammar, R., Mohamed, M. E., Alfwuaires, M., Abdulaziz Alamer, S., Bani Ismail, M., Veeraraghavan, V. P., Sekar, A. K., Ksouri, R., & Rajendran, P. (2022). Anti-Inflammatory Activity of Geraniol Isolated from Lemon Grass on Ox-LDL-Stimulated Endothelial Cells by Upregulation of Heme Oxygenase-1 via PI3K/Akt and Nrf-2 Signaling Pathways. Nutrients, 14(22). https://doi.org/10.3390/nu14224817
Borges, R. S., Ortiz, B. L. S., Pereira, A. C. M., Keita, H., & Carvalho, J. C. T. (2019). Rosmarinus officinalis essential oil: A review of its phytochemistry, anti-inflammatory activity, and mechanisms of action involved. J Ethnopharmacol, 229, 29-45. https://doi.org/10.1016/j.jep.2018.09.038
Boutilier, A. J., & Elsawa, S. F. (2021). Macrophage Polarization States in the Tumor Microenvironment. Int J Mol Sci, 22(13). https://doi.org/10.3390/ijms22136995
Cinelli, M. A., Do, H. T., Miley, G. P., & Silverman, R. B. (2020). Inducible nitric oxide synthase: Regulation, structure, and inhibition. Med Res Rev, 40(1), 158-189. https://doi.org/10.1002/med.21599
Dangol, S., Poudel, D. K., Ojha, P. K., Maharjan, S., Poudel, A., Satyal, R., Rokaya, A., Timsina, S., Dosoky, N. S., Satyal, P., & Setzer, W. N. (2023). Essential Oil Composition Analysis of Cymbopogon Species from Eastern Nepal by GC-MS and Chiral GC-MS, and Antimicrobial Activity of Some Major Compounds. Molecules, 28(2). https://doi.org/10.3390/molecules28020543
Gemeda, N., Tadele, A., Lemma, H., Girma, B., Addis, G., Tesfaye, B., Abebe, A., Gemechu, W., Yirsaw, K., Teka, F., Haile, C., Amano, A., Woldkidan, S., Geleta, B., & Debella, A. (2018). Development, Characterization, and Evaluation of Novel Broad-Spectrum Antimicrobial Topical Formulations from Cymbopogon martini (Roxb.) W. Watson Essential Oil. Evid Based Complement Alternat Med, 2018, 9812093. https://doi.org/10.1155/2018/9812093
Guo, L., Morin, E. E., Yu, M., Mei, L., Fawaz, M. V., Wang, Q., Yuan, Y., Zhan, C. G., Standiford, T. J., Schwendeman, A., & Li, X. A. (2022). Replenishing HDL with synthetic HDL has multiple protective effects against sepsis in mice. Sci Signal, 15(725), eabl9322. https://doi.org/10.1126/scisignal.abl9322
Haro-González, J. N., Castillo-Herrera, G. A., Martínez-Velázquez, M., & Espinosa-Andrews, H. (2021). Clove Essential Oil (Syzygium aromaticum L. Myrtaceae): Extraction, Chemical Composition, Food Applications, and Essential Bioactivity for Human Health. Molecules, 26(21). https://doi.org/10.3390/molecules26216387
Kalagatur, N. K., Nirmal Ghosh, O. S., Sundararaj, N., & Mudili, V. (2018). Antifungal Activity of Chitosan Nanoparticles Encapsulated With Cymbopogon martinii Essential Oil on Plant Pathogenic Fungi Fusarium graminearum. Front Pharmacol, 9, 610. https://doi.org/10.3389/fphar.2018.00610
Kang, Z. P., Wang, M. X., Wu, T. T., Liu, D. Y., Wang, H. Y., Long, J., Zhao, H. M., & Zhong, Y. B. (2021). Curcumin Alleviated Dextran Sulfate Sodium-Induced Colitis by Regulating M1/M2 Macrophage Polarization and TLRs Signaling Pathway. Evid Based Complement Alternat Med, 2021, 3334994. https://doi.org/10.1155/2021/3334994
Kishore, A., & Petrek, M. (2021). Roles of Macrophage Polarization and Macrophage-Derived miRNAs in Pulmonary Fibrosis. Front Immunol, 12, 678457. https://doi.org/10.3389/fimmu.2021.678457
Le, Y., Cao, W., Zhou, L., Fan, X., Liu, Q., Liu, F., Gai, X., Chang, C., Xiong, J., Rao, Y., Li, A., Xu, W., Liu, B., Wang, T., Wang, B., & Sun, Y. (2020). Infection of Mycobacterium tuberculosis Promotes Both M1/M2 Polarization and MMP Production in Cigarette Smoke-Exposed Macrophages. Front Immunol, 11, 1902. https://doi.org/10.3389/fimmu.2020.01902
Linghu, K. G., Wu, G. P., Fu, L. Y., Yang, H., Li, H. Z., Chen, Y., Yu, H., Tao, L., & Shen, X. C. (2019). 1,8-Cineole Ameliorates LPS-Induced Vascular Endothelium Dysfunction in Mice via PPAR-γ Dependent Regulation of NF-κB. Front Pharmacol, 10, 178. https://doi.org/10.3389/fphar.2019.00178
Liu, L., Guo, H., Song, A., Huang, J., Zhang, Y., Jin, S., Li, S., Zhang, L., Yang, C., & Yang, P. (2020). Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways. BMC Immunol, 21(1), 32. https://doi.org/10.1186/s12865-020-00355-y
Margraf, A., & Perretti, M. (2022). Immune Cell Plasticity in Inflammation: Insights into Description and Regulation of Immune Cell Phenotypes. Cells, 11(11). https://doi.org/10.3390/cells11111824
Poulin, J., Paulocik, C., & Veall, M. A. (2022). Found in the Folds: A Rediscovery of Ancient Egyptian Pleated Textiles and the Analysis of Carbohydrate Coatings. Molecules, 27(13). https://doi.org/10.3390/molecules27134103
Roe, K. (2021). An inflammation classification system using cytokine parameters. Scand J Immunol, 93(2), e12970. https://doi.org/10.1111/sji.12970
Scotti, R., Stringaro, A., Nicolini, L., Zanellato, M., Boccia, P., Maggi, F., & Gabbianelli, R. (2021). Effects of Essential Oils from Cymbopogon spp. and Cinnamomum verum on Biofilm and Virulence Properties of Escherichia coli O157:H7. Antibiotics (Basel), 10(2). https://doi.org/10.3390/antibiotics10020113
Skopelja-Gardner, S., Tai, J., Sun, X., Tanaka, L., Kuchenbecker, J. A., Snyder, J. M., Kubes, P., Mustelin, T., & Elkon, K. B. (2021). Acute skin exposure to ultraviolet light triggers neutrophil-mediated kidney inflammation. Proc Natl Acad Sci U S A, 118(3). https://doi.org/10.1073/pnas.2019097118
Tran, N., Garcia, T., Aniqa, M., Ali, S., Ally, A., & Nauli, S. M. (2022). Endothelial Nitric Oxide Synthase (eNOS) and the Cardiovascular System: in Physiology and in Disease States. Am J Biomed Sci Res, 15(2), 153-177.
van Os, N., Javed, A., Broere, F., van Dijk, A., Balhuizen, M. D., van Eijk, M., Rooijakkers, S. H. M., Bardoel, B. W., Heesterbeek, D. A. C., Haagsman, H. P., & Veldhuizen, E. (2022). Novel insights in antimicrobial and immunomodulatory mechanisms of action of PepBiotics CR-163 and CR-172. J Glob Antimicrob Resist, 30, 406-413. https://doi.org/10.1016/j.jgar.2022.07.009
Wang, J., Su, B., Zhu, H., Chen, C., & Zhao, G. (2016). Protective effect of geraniol inhibits inflammatory response, oxidative stress and apoptosis in traumatic injury of the spinal cord through modulation of NF-κB and p38 MAPK. Exp Ther Med, 12(6), 3607-3613. https://doi.org/10.3892/etm.2016.3850
Wu, Y. X., Jiang, F. J., Liu, G., Wang, Y. Y., Gao, Z. Q., Jin, S. H., Nie, Y. J., Chen, D., Chen, J. L., & Pang, Q. F. (2021). Dehydrocostus Lactone Attenuates Methicillin-Resistant Staphylococcus aureus-Induced Inflammation and Acute Lung Injury via Modulating Macrophage Polarization. Int J Mol Sci, 22(18). https://doi.org/10.3390/ijms22189754
Yin, C., Liu, B., Wang, P., Li, X., Li, Y., Zheng, X., Tai, Y., Wang, C., & Liu, B. (2020). Eucalyptol alleviates inflammation and pain responses in a mouse model of gout arthritis. Br J Pharmacol, 177(9), 2042-2057. https://doi.org/10.1111/bph.14967
Zhou, X., Chen, Y., Cui, L., Shi, Y., & Guo, C. (2022). Advances in the pathogenesis of psoriasis: from keratinocyte perspective. Cell Death Dis, 13(1), 81. https://doi.org/10.1038/s41419-022-04523-3
Zhu, L. J., Li, F., & Zhu, D. Y. (2023). nNOS and Neurological, Neuropsychiatric Disorders: A 20-Year Story. Neurosci Bull, 1-15. https://doi.org/10.1007/s12264-023-01060-7