臺灣博碩士論文加值系統

近年來多數國家人口逐漸進入高齡化社會,具有抗老化活性之藥妝產品漸漸受到消費者的注意,決定皮膚光老化的主要原因之一是膠 原蛋白,而基質金屬蛋白酶-1 (matrix metalloproteinase, MMP-1) 會影 響膠原蛋白的降解,因此 MMP-1 在皮膚抗光老化的作用扮演重要的 角色。臺灣火刺木 ( Pyracantha koidzumii (Hanyata) Rehder) 為薔薇科 (Rosaceae) 火刺木屬 (Pyracantha) 之常綠灌木,屬於臺灣特有種植物。過去研究指出臺灣火刺木具有美白作用,本研究以其果實之 95% 乙醇萃取物進行部分劃分,利用活性追蹤方式探討在 WS-1 細胞之抗 光老化作用,結果顯示粗萃物、乙酸乙酯、正丁醇及水層之萃取物對 細胞存活率影響不大,且乙酸乙酯層萃取物具較明顯抑制 MMP-1 活 性。將乙酸乙酯層萃取物進行藥物奈米劑型的探討,得到奈米等級 (粒徑= 148.3 nm)、穩定 (多分散性指數= 0.131) 且包覆率為 90% 之 藥物劑型。進一步從乙酸乙酯層萃取物進行成分分離純化,經理化及 光譜相關數據與參考文獻比對得到三個化合物:分別為 PK1, PK2 及 PK3。在人類纖維母細胞 (WS-1) 中具有抑制 MMP-1 之活性,且 PK2 和 PK3 具有細胞內抗氧化的作用。期望透過本研究在未來對臺灣火 刺木發展抗光老化及奈米劑型有良好貢獻。

In recent years, most countries have gradually entered the ageing society, so cosmeceuticals that possess anti-photoaging activities have attracting more and more attention. Matrix metalloprpteinase-1 (MMP-1) affects the degradation of collagen, which is one of the main cause of skin photoaging. Therefore MMP-1 inhibitor plays an important role in anti-photoaging of the skin. Pyracantha koidzumii (Hanyata) Rehder (Rosaceae), an evergreen, is one of Taiwanese endemic plants. Previous study indicated that P. koidzumii has whitening activity. In this study, fruits of P. koidzumii were extracted with 95% ethanol and then partitioned with a sequence of n-hexane, ethyl acetate (EA), and n-butanol (n-BuOH). We evaluated the activities of the extracts and partitioned fractions in human fibroblast (WS-1). The results showed that EA fraction significantly decreased the MMP-1 activity in WS-1 cells. The EA fraction was performed to explore the pharmaceutical dosage form of fraction. We acquired the nano level (particle size= 148.3 nm) and stable (polydispersity index= 0.131) dosage form. After bio-guided column chromatography, we obtain three known compounds. The structures of these compounds were identified as PK1, PK2 and PK3 by comparing with the physical and spectroscopic characterizations. Among these compounds, PK2 significantly decreased the MMP-1 activity in WS-1 cells. PK2 and PK3 significantly decrease the intracellular reactive oxygen species (ROS) contents in WS-1 cells. The present results imply that P. koidzumii and the EA fraction nanometer particles may be useful in anti-photoaging design in the future.

目錄 目錄...................................................................................................... i
圖目錄................................................................................................ iv 表目錄..................................................................................................v 縮寫表................................................................................................ vi 摘要.................................................................................................. viii Abstract .............................................................................................. ix 第一章 緒論........................................................................................1 一、 植物介紹...................................................................................1
(一) 臺灣火刺木簡介........................................................................1 (二) 火刺木屬植物相關介紹............................................................1 (三) 火刺木屬植物活性研究回顧....................................................4 (四) 火刺木屬植物之化學成分研究................................................5
二、 皮膚光老化之研究.................................................................18 (一) 皮膚結構介紹..........................................................................18 (二) 紫外線對皮膚之影響..............................................................19 (三) 光老化之機制..........................................................................20
三、 皮膚相關外用製劑介紹.........................................................22 (一) 化妝品劑型介紹......................................................................22 (二) 藥物傳遞系統..........................................................................23 (三) 奈米劑型之相關參數..............................................................24
四、 研究動機.................................................................................26 第二章 實驗材料與方法..................................................................27 一、 臺灣火刺木之萃取與分離.....................................................27 (一) 一般試藥及溶媒......................................................................27 (二) 色層分析法材料......................................................................27 (三) 儀器..........................................................................................28 (四) 臺灣火刺木萃取及分離流程..................................................29 (五) 各成分之物理數據..................................................................30
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二、 臺灣火刺木之活性實驗.........................................................32 (一) 藥品及試劑..............................................................................32 (二) 儀器..........................................................................................32 (三) 人類皮膚纖維母細胞 (WS-1) 細胞培養 .............................33 (四) 人類皮膚纖維母細胞 (WS-1) 細胞存活率試驗.................33 (五) 人類皮膚纖維母細胞 (WS-1) 細胞內 MMP-1 活性試驗...34 (六) 人類皮膚纖維母細胞 (WS-1) 細胞內抗氧化活性試驗.....34
三、 統計方法.................................................................................35 四、 臺灣火刺木萃取物奈米劑型製備.........................................36 (一) 奈米製備之試劑及儀器..........................................................36 (二) 奈米製備之方法......................................................................36 (三) 奈米參數測定..........................................................................37 第三章 結果......................................................................................38 一、 臺灣火刺木之活性探討.........................................................38 (一) 植物部分劃分萃取物於WS-1細胞存活率試驗..................38 (二) 植物部分劃分萃取物於 UVB 照射 WS-1 細胞存活率試驗40 (三) 植物部分劃分萃取物於 WS-1 細胞內 MMP-1 活性試驗 ...42 二、 臺灣火刺木活性萃取物之奈米劑型.....................................44 (一) 臺灣火刺木乙酸乙酯層萃取物於HPLC之定量分析.........44 (二) 臺灣火刺木乙酸乙酯層萃取物於奈米劑型之分析.............46
(三) 臺灣火刺木乙酸乙酯層萃取物於奈米劑型之細胞存活率試
驗 ......................................................................................................47 三、 臺灣火刺木化合物之結構鑑定.............................................49 (一) PK1 之結構解析......................................................................49 (二) PK2 之結構解析......................................................................51 (三) PK3 之結構解析 ......................................................................53 四、 臺灣火刺木化合物之活性探討.............................................55 (一) 分離化合物於WS-1細胞存活率試驗..................................55 (二) 分離化合物於 UVB 照射 WS-1 細胞存活率試驗................57
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(三) 分離化合物於 WS-1 細胞內 MMP-1 活性試驗 ...................59
(四) 分離化合物於WS-1細胞內抗氧化活性試驗......................61 第四章 討論......................................................................................63 第五章 結論......................................................................................66 第六章 參考文獻..............................................................................67 第七章 附錄......................................................................................73
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圖目錄
Figure 1. The sun UV irradiation damage to skin connective tissue ........20 Figure 2. Cell viabilities of partitioned fractions from Pyracantha koidzumii in WS-1 cells ............................................................................39 Figure 3. Cell viabilities of partitioned fractions from Pyracantha koidzumii with UVB irradiation in WS-1 cells.........................................41 Figure 4. MMP-1 contents of partitioned fractions from Pyracantha koidzumii with UVB irradiation in WS-1 cells.........................................43 Figure 5. The fluorescence intensity of EA-layer by HPLC ....................44 Figure 6. Cell viabilities of EA fraction nanometer particles in WS-1 cells ...................................................................................................................48 Figure 7. Cell viabilities of PK1-3 from Pyracantha koidzumii in WS-1 cells ...........................................................................................................56 Figure 8. Cell viabilities of PK1-3 from Pyracantha koidzumii with UVB irradiation in WS-1 cells ...........................................................................58 Figure 9. MMP-1 contents of PK1-3 from Pyracantha koidzumii with UVB irradiation in WS-1 cells..................................................................60 Figure 10. ROS contents of PK1-3 from Pyracantha koidzumii with UVB irradiation in WS-1 cells ...........................................................................62 Figure 11. Scheme of Pyracantha koidzumii extraction and compounds. ...................................................................................................................73 Figure 12. 1H NMR (Methanol-d4, 500 MHz) spectrum of PK1 .............74 Figure 13. 1H NMR (Methanol-d4, 300 MHz) spectrum of PK2 .............75 Figure 14. 1H NMR (Methanol-d4, 500 MHz) spectrum of PK3 .............76 Figure 15. 13C NMR (Methanol-d4, 125 MHz) spectrum of PK3 ............77
iv
表目錄
Table 1. Carotenoids in Pyracantha species...............................................6 Table 2. Biphenyl derivatives in Pyracantha species.................................7 Table 3. Dibenzofurans in Pyracantha species...........................................8 Table 4. Flavonoids in Pyracantha species ..............................................10 Table 5. Glycosides in Pyracantha species ..............................................14 Table 6. Steroids in Pyracantha species ...................................................16 Table 7. Triterpenoids in Pyracantha species...........................................16 Table 8. Others in Pyracantha species .....................................................17 Table 9. Standard curve of P. koidzumii EA-nano ....................................45 Table 10. The parameter of EA fraction nanometer particles ...................46 Table 11. 500 MHz 1H-NMR spectral data of PK1 (δ values, in MeOH-d4 J in Hz)......................................................................................................50 Table 12. 300 MHz 1H-NMR spectral data of PK2 (δ values, in MeOH-d4, J in Hz)......................................................................................................52 Table 13. 500 MHz 1H-NMR and 125 MHz 13C NMR spectral data of PK3 (δ values, in MeOH-d4, J in Hz) .......................................................54

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