本實驗評估鑫大埔生技實業股份有限公司之牛樟芝A樣本、牛樟芝B樣本及洛神花萃取物之清除自由基能力、對人類皮膚纖維母細胞CCD-966SK之短期細胞毒性、細胞內活性氧清除力以及對於傷口修復及酪胺酸酶誘導黑色素生成利用樣品抑制之影響。DPPH自由基清除試驗結果，洛神花萃取液1000g/ml之清除率為80%，牛樟芝B樣本5000g/ml自由基清除率為75%，牛樟芝A樣本10g/ml自由基清除率為23%。細胞培養試驗系統中，MTT試驗評估牛樟芝A樣本、牛樟芝B樣本及洛神花萃取液樣本72小時處理下保有90%以上細胞存活之濃度分別為100g/ml、100g/ml及1000g/ml。傷口癒合實驗結果，牛樟芝A樣本添加至較高濃度100g/ml處理12-36小時細胞都無生長遷移現象；牛樟芝B樣本在100g/ml以及洛神花萃取液100g/ml及500g/ml濃度處理12小時，傷口開始出現癒合，至36小時已接近100%癒合，控制組在36小時之傷口修復程度約40%。酪胺酸酶抑制實驗結果，牛樟芝A樣本濃度添加至50g/ml黑色素抑制率為56%，牛樟芝B樣本最高濃度500g/ml黑色素抑制率為50%，洛神花萃取液隨著濃度提升至2000g/ml黑色素抑制率為64%，其中洛神花萃取液能夠有效的抑制黑色素生成。細胞氧化壓力實驗結果，ROS有效抑制濃度牛樟芝A樣本1g/ml、牛樟芝B樣本500g/ml、洛神花萃取液2000g/ml，抑制率分別為36%、49%、62%。

In this study, we evaluated the short-term cytotoxicity, intracellular reactive oxygen scavenging capacity, wound repairing, tyrosinase-induced melanogenesis and scavenging free radical scavenging ability of the human skin fibroblasts CCD-966SK which treated with Antrodia cinnamomea sample A (ACA), Antrodia cinnamomea sample B (ACB) and extraction of Hibiscus sabdariffa (EHS). The results of DPPH free radical scavenging assay, The clearance rate of EHS (1000 g/ml), ACB (5000 g/ml) and ACA (10 g/ml) were 80%, 75% and 23% respectively. For MTT assay, the survival rate of CCD-966SK cell were 90% when treated with ACA (100 g/ml), ACB (100 g/ml) and EHS (1000 g/ml) for 72 hours. For wound healing assay of each treatments, treated with ACA (100 g/ml) haven’t shown cell growth and migration at 12-48 hours, but treated with ACB (100 g/ml) and EHS (100 g/ml and 500 g/ml) shown wound healing. Moreover, the wound healing rate were reached 100% at 36 hours which negitive control only reached 40%. For tyrosinase inhibition assay, the melanin inhibition rate of ACA (50 g/ml), ACB (500 g/ml) and EHS (2000 g/ml) were 56%, 50% and 64%, respectively. The EHS shown most effect melanin inhibition rate compare to other groups. For Cell oxidative stress assay, the ROS inhibition rate of ACA (1 g/ml), ACB (500 g/ml) and EHS (2000 g/ml) were 36%, 49% and 62%, respectively.

中文摘要 I
Abstract II
誌謝 IV
目錄 V
第一章 前言 1
1.1 研究背景 1
1.2 研究動機與架構 2
1.3 研究成果的重要性 2
第二章 文獻回顧 3
2.1牛樟芝的功能性以及成分 3
2.2洛神花的功能性以及成份 4
2.3氧化壓力的形成 5
2.4抗氧化物與自由基在身體中的作用 6
2.5 酪胺酸酶形成黑色素 7
2.6 傷口癒合的形成 8
第三章 材料與方法 9
3.1 實驗材料 9
3.1.1 儀器設備 9
3.1.2 試劑及緩衝溶液 10
3.1.3 細胞品系 11
3.2 實驗方法 11
3.3牛樟芝及洛神花萃取液 11
3.4細胞培養 11
3.4.1 Dulbecco’s Modified Eagle’s medium配製 11
3.4.2細胞繼代 12
3.4.3冷凍細胞 12
3.4.4解凍細胞 13
3.4.5細胞計數 13
3.5細胞毒性試驗 13
3.6抗氧化試驗 14
3.7傷口癒合試驗 14
3.8細胞氧化壓力試驗 15
3.9酪胺酸酶抑制試驗 15
第四章 結果 17
4.1細胞毒性試驗 17
4.2抗氧化試驗 17
4.3傷口癒合試驗 18
4.4細胞氧化壓力試驗 18
4.5酪胺酸酶抑制試驗 19
第五章 討論 30
5.1細胞毒性試驗 30
5.2抗氧化試驗 30
5.3傷口癒合試驗 31
5.4細胞氧化壓力試驗 31
5.5酪胺酸酶抑制試驗 32
參考文獻 33

Ajiboye, Taofeek O, Salawu, Nasir A, Yakubu, Musa T, Oladiji, Adenike T, Akanji, Musbau A, & Okogun, Joseph I. (2011). Antioxidant and drug detoxification potentials of Hibiscus sabdariffa anthocyanin extract. Drug and chemical toxicology, 34(2), 109-115.
Ali, Badreldin H, Wabel, Naser Al, & Blunden, Gerald. (2005). Phytochemical, pharmacological and toxicological aspects of Hibiscus sabdariffa L.: a review. Phytotherapy research, 19(5), 369-375.
Aranda, A, Sequedo, L, Tolosa, L, Quintas, G, Burello, E, Castell, JV, & Gombau, L. (2013). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: a quantitative method for oxidative stress assessment of nanoparticle-treated cells. Toxicology in Vitro, 27(2), 954-963.
Brown, Lawrence F, Yeo, KiangTeck, Berse, Brygida, Yeo, Tet-Kin, Senger, Donald R, Dvorak, Harold F, & Van De Water, Livingston. (1992). Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. Journal of Experimental Medicine, 176(5), 1375-1379.
Carmona-Jiménez, Yolanda, García-Moreno, M Valme, Igartuburu, Jose M, & Barroso, Carmelo Garcia. (2014). Simplification of the DPPH assay for estimating the antioxidant activity of wine and wine by-products. Food chemistry, 165, 198-204.
Chan, Godfrey Chi-Fung, Chan, Wing Keung, & Sze, Daniel Man-Yuen. (2009). The effects of β-glucan on human immune and cancer cells. Journal of hematology & oncology, 2(1), 25.
Chang, TT, & Chou, WN. (1995). Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan. Mycological Research, 99(6), 756-758.
Chien, Chih-Ching, Tsai, Mei-Ling, Chen, Chin-Chu, Chang, Shing-Jyh, & Tseng, Ching-Hui. (2008). Effects on tyrosinase activity by the extracts of Ganoderma lucidum and related mushrooms. Mycopathologia, 166(2), 117.
de Souza, Russell J, Mente, Andrew, Maroleanu, Adriana, Cozma, Adrian I, Ha, Vanessa, Kishibe, Teruko, . . . Beyene, Joseph. (2015). Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies. Bmj, 351, h3978.
Hseu, You-Cheng, Chang, Weng-Cheng, Hseu, Yi-Ting, Lee, Chia-Ying, Yech, Yi-Jen, Chen, Pei-Chun, . . . Yang, Hsin-Ling. (2002). Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life sciences, 71(4), 469-482.
Jung, Sung-Won, Lee, Nam-Kyung, Kim, Seok-Joong, & Han, Dae-Seok. (1995). Screening of tyrosinase inhibitor from plants. Korean Journal of Food Science and Technology, 27(6), 891-896.
Liou, Geou-Yarh, & Storz, Peter. (2010). Reactive oxygen species in cancer. Free radical research, 44(5), 479-496.
Lobo, Vijaya, Patil, Avinash, Phatak, A, & Chandra, Naresh. (2010). Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy reviews, 4(8), 118.
Martin, Paul. (1997). Wound healing--aiming for perfect skin regeneration. Science, 276(5309), 75-81.
Mau, Jeng-Leun, Huang, Pei-Ning, Huang, Shih-Jeng, & Chen, Chin-Chu. (2004). Antioxidant properties of methanolic extracts from two kinds of Antrodia camphorata mycelia. Food chemistry, 86(1), 25-31.
Mohd-Esa, Norhaizan, Hern, Fong Shin, Ismail, Amin, & Yee, Chew Lye. (2010). Antioxidant activity in different parts of roselle (Hibiscus sabdariffa L.) extracts and potential exploitation of the seeds. Food Chemistry, 122(4), 1055-1060.
Muhammad, TB, & Shakib, AB. (1995). Jus hibiscus: Bukan sekadar minuman biasa. Dewan Ekonomi, 12-14.
Nihei, Ken-ichi, & Kubo, Isao. (2003). Identification of oxidation product of arbutin in mushroom tyrosinase assay system. Bioorganic & medicinal chemistry letters, 13(14), 2409-2412.
Omotani, Hidetoshi, Yasuda, Makoto, Ishii, Ritsuko, Ikarashi, Tsukasa, Fukuuchi, Tomoko, Yamaoka, Noriko, . . . Nakagomi, Kazuya. (2016). Analysis of l-DOPA-derived melanin and a novel degradation product formed under alkaline conditions. Journal of pharmaceutical and biomedical analysis, 125, 22-26.
Saglio, Giuseppe, Kim, Dong-Wook, Issaragrisil, Surapol, Le Coutre, Philipp, Etienne, Gabriel, Lobo, Clarisse, . . . Hughes, Timothy P. (2010). Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia. New England Journal of Medicine, 362(24), 2251-2259.
Siegrist, Walter, & Eberle, Alex N. (1986). In situ melanin assay for MSH using mouse B16 melanoma cells in culture. Analytical biochemistry, 159(1), 191-197.
Tolulope, Mary. (2007). Cytotoxicity and antibacterial activity of methanolic extract of Hibiscus sabdariffa. Journal of Medicinal Plants Research, 1(1), 009-013.
Tsai, Ming-Chi, Song, Tuzz-Ying, Shih, Ping-Hsiao, & Yen, Gow-Chin. (2007). Antioxidant properties of water-soluble polysaccharides from Antrodia cinnamomea in submerged culture. Food Chemistry, 104(3), 1115-1122.
Tsai, Pi-Jen, McIntosh, John, Pearce, Philip, Camden, Blake, & Jordan, Brian R. (2002). Anthocyanin and antioxidant capacity in Roselle (Hibiscus sabdariffa L.) extract. Food research international, 35(4), 351-356.
Tsai, PJ, & Ou, ASM. (1996). Colour degradation of dried roselle during storage. Food Sci, 23, 629-640.
柳爱莲, & 刘绣华. (2007). 天然产物抗 DPPH 自由基活性研究. 周口師範學院學報, 24(5), 80-82.