臺灣博碩士論文加值系統

樟芝(Antrodia camphorata)是台灣發現的特有物種，已知它是一種藥用菇類，廣泛應用於傳統中藥上。它的生理活性物質尚在分析中，目前已知主要的有效成分為多醣體及三萜類。本研究的目的在探討以樟芝菌絲體深層培養所獲得的胞外多醣體，應用於抑制3T3纖維母細胞所分泌基質金屬蛋白酶(matrix metalloproteinases, MMP)的活性；經由gelatin-based zymography進行 MMP-2及MMP-9的活性分析，我們發現樟芝胞外多醣體於0.5 mg/ml的處理劑量下，可以抑制MMP-2及MMP-9的活性，於48小時最為顯著。另一方面，利用不同的酒精濃度沉澱樟芝胞外多醣體，經由膠體過濾層析法(gel permeation chromatography)分析，證實不同的酒精濃度可分離得到不同分子量的多醣體；這些不同分子量的多醣體中，以33% 酒精濃度沉澱分離的多醣體抑制MMP-9的活性最顯著，而50~75% 沉澱酒精濃度所分離的多醣體抑制MMP-2活性的效果最佳。令人驚訝的是50~75%酒精濃度沉澱所得到的多醣體，於0.5 mg/ml的劑量，處理纖維母細胞4~8小時，在與控制組比較之下，發現膠原蛋白的累積量可增加38%。因此，我們認為樟芝胞外多醣體可應用於抗老化化妝品的成份。

Antrodia camphorata, an endemic species in Taiwan, is known as a medicinal mushroom and currently used as a traditional Chinese medicine. The constituent and bioactivity of A. camphorata is still in the analysis. For the present it is well known that polysaccharides and triterpenoids are major effective components in this medicinal fungus. The purpose of this study was to investigate the capacities of exopolysaccharides extracted from mycelium culture of A. camphorata in inhibiting the activities of matrix metalloproteinases (MMPs) secreted from fibroblasts. The analyses of gelatin-based zymography reveal that A. camphorata exopolysaccharides do suppress the activities of MMP-2 (gelatinase A, EC 3.4.24.24) and MMP-9 (gelatinase B, EC 3.4.24.35) at the treated concentration of 0.5 mg/ml. On the other hand, we undertook various concentrations of ethanol precipitation with A. camphorata exopolysaccharides to obtain several fractions of polysaccharides with different molecular weights determined by gel permeation chromatography. The different fractions of polysaccharides were also applied to evaluate the inhibitory ability on the activities of MMP-2 and MMP-9. The results presented here show that 33% ethanol-precipitated exopolysaccharides exhibit the markedly repressive ability on the activity of MMP-9, whereas 50~75% ethanol-precipitated exopolysaccharides significantly inhibit MMP-2 activity. Surprisingly, we also found that 50~75% ethanol-precipitated exopolysaccharides could give rise to collagen accumulation by 38% in the culture medium of fibroblasts when concentration was set at 0.5 mg/ml. Therefore, we suggest that exopolysaccharides from A. camphorata may show the antiaging capacities and will be an effective ingredient for utilization in antiaging cosmetic formulations.

目錄

中文摘要.......................................................................................................... I
英文摘要...................................................................................................II、III
誌謝.................................................................................................................IV
表目錄.............................................................................................................IX
圖目錄 ............................................................................................................IX
縮寫表.............................................................................................................XII

第一章、緒論.......................................................................................................1
一、文獻回顧...................................................................................................1
(一) 前言.......................................................................................................1
1. 樟芝簡介...............................................................................................1 2. 樟芝的分類和特徵...............................................................................2
3. 樟芝的活性成分及弁?......................................................................5
4. 生物化妝品之研究...............................................................................6
(二) 老化與皮膚之間的關係.......................................................................6
1.人體皮膚的構造...................................................................6
2. 膠原蛋白..............................................................................................10
3. 基質金屬蛋白酶(MMPs).....................................................................12
4. 皮膚與老化的關係..............................................................................14
二、研究動機....................................................................................................16

第二章、材料與方法.......................................................................................... 17
一、材料與藥品............................................................................................... 17
1. 材料.........................................................................................................17
1.1 樟芝菌株及細胞來源.....................................................................17
1.2 培養基組成.....................................................................................18
1.3 細胞培養與細胞測試劑.................................................................18
1.4 儀器設備.........................................................................................19
1.5 緩衝液與溶液配製........................................................................ 20
二、實驗方法.......................................................................................................21
1. 樟芝菌種培養方法.................................................................................21
1.1 固態培養............................................................................................21
1.2 樟芝菌絲體液態培養.......................................................................21
2. 多醣體萃取方法.....................................................................................21
3. 總多醣體檢測.........................................................................................22
4. 階梯式酒精沉澱法.................................................................................22
5. 膠體過濾層析分析分子量的分佈.........................................................25
6. 細胞培養.................................................................................................25
6.1 細胞繼代培養及分配....................................................................26
6.2 細胞存活率試驗 (MTT assay)......................................................26
7. 電泳酵素分析法 (gelatin-based zymography) ......................................27
7.1 Separating gel的製備......................................................................27
7.2 Stacking gel的製備.........................................................................27
7.3電泳條件..........................................................................................28
7.4 SDS聚丙烯胺凝膠膠片反應..........................................................28
7.5 SDS聚丙烯胺凝膠膠片染色..........................................................28
8. 酒精濃度為( 50~75% )沉澱多醣體的方法...........................................29
9. 酵素分泌試驗 .......................................................................................29
10. 膠原蛋白濃度分析...............................................................................30
11. 皮膚的弁鄔妠��?..............................................................................30
第三章、結果......................................................................................................31
1. 樟芝發酵液胞外總多醣體的濃度分析.................................................31
2. 以膠體穿透層析法(Gel Permeation Chromatography)分析樟芝胞外多醣體的分子量分佈................................................................................31
3. 樟芝胞外多醣體的細胞毒性分析.........................................................33
4. 樟芝胞外總多醣體對於MMPs的活性分析.........................................34
5. 不同酒精濃度範圍所沉澱分離的樟芝胞外多醣體存在下對於MMPs的活性分析.............................................................................................35
6. 50~75% 酒精濃度範圍所沉澱分離的樟芝胞外多醣體對於3T3細胞的細胞毒性分析....................................................................................36
7. 50~75% 酒精濃度範圍所沉澱分離的樟芝胞外多醣體對於3T3細胞的MMPs酵素分泌分析........................................................................37
8. 50~75% 酒精濃度範圍所沉澱分離的樟芝胞外多醣體對於3T3細胞培養基內膠原蛋白含量的分析............................................................37
9. 樟芝胞外總多醣體所調製凝膠化妝品對於人體皮膚的弁鄔妠��?38

第四章、討論............................................................................................................40
第五章、結論.............................................................................................................43
參考文獻....................................................................................................................44






附錄
［表目錄］
表一、樟芝胞外多醣體於培養基內的濃度(以95% 酒精濃度進行沉澱)...50表二、沉澱10個fractions的樟芝胞外多醣所利用的不同酒精濃度範圍...51
表三、膠體穿透層析法(Gel Permeation Chromatography)所使用的多糖體標準品之分子量(Molecular Weight, MW)、滯留時間(Retention Time, RT) 及標準品分子量的log[log(MW)]................................................51
表四、於不同酒精濃度範圍所沉澱分離10個fractions的樟芝胞外多醣體經由膠體穿透層析法分析所獲得的滯留時間與分子量分佈的關係..........................................................................................................58
表五、樟芝胞外多醣體所調製凝膠化妝品對於人體皮膚的弁鄔妗��?.........................................................................................................70
［圖目錄］
圖一、 以不同濃度的葡萄糖溶液作為標準樣品及其在485 nm的吸光值所作出標準回歸線。.............................................................................50
圖二、膠體穿透層析法所使用的多糖體標準品(來自Polyethylene Oxide Aqueous Calibration Kit)之滯留時間(Retention Time)及標準品分子量的log值[log (molecular weight)]所作之標準曲線。....................52
圖三、Fraction 1樟芝胞外多醣體之膠體穿透層析法之層析圖.................53
圖四、Fraction 2樟芝胞外多醣體之膠體穿透層析法之層析圖..................53
圖五、Fraction 3樟芝胞外多醣體之膠體穿透層析法之層析圖..................54
圖六、Fraction 4樟芝胞外多醣體之膠體穿透層析法之層析圖..................54
圖七、Fraction 5樟芝胞外多醣體之膠體穿透層析法之層析圖.................55
圖八、Fraction 6樟芝胞外多醣體之膠體穿透層析法之層析圖...................55
圖九、Fraction 7樟芝胞外多醣體之膠體穿透層析法之層析圖..................56
圖十、Fraction 8樟芝胞外多醣體之膠體穿透層析法之層析圖..................56
圖十一、Fraction 9樟芝胞外多醣體之膠體穿透層析法之層析圖..............57
圖十二、Fraction 10樟芝胞外多醣體之膠體穿透層析法之層析圖.............57
圖十三、不同濃度(0~1.0 mg/ml)的樟芝胞外多醣體處理3T3細胞2天，檢測3T3細胞存活率。.....................................................................59
圖十四、樟芝胞外多醣體以0.5 mg/ml的劑量處理不同天數(1~3天)，檢測3T3細胞存活率。.......................................................................60
圖十五、以0.5 mg/ml的樟芝胞外多醣體處理3T3細胞培養基，反應不同時間(0~120小時)，再進行MMPs的活性分析。.........................61
圖十六、以不同濃度(0~0.5 mg/ml)的樟芝胞外多醣體處理3T3細胞培養基，反應48小時，再進行MMPs的活性分析。..............................62
圖十七、分別利用Fractions 1~5的樟芝胞外多醣體處理3T3細胞培養基，以0.5 mg /ml的劑量，反應48小時，再檢測MMPs的活性。........63
圖十八、分別利用Fractions 6~10的樟芝胞外多醣體處理3T3細胞培養基，以0.5 mg /ml的劑量，反應48小時，檢測MMPs的活性。.................................................................................................64
圖十九、50~75% 酒精濃度範圍所沉澱分離的樟芝胞外多醣體，以0.5 mg/ml的劑量處理不同天數(1~3天)，再進行MTT assay，以分析3T3細胞的存活率。...................................................................65
圖二十、50~75%酒精濃度範圍所沉澱分離的樟芝胞外多醣體，以1.0 mg/ml的劑量處理不同天數(1~3天)，再進行MTT assay，以分析3T3細胞的存活率。..................................................................66
圖二十一、50~75% 酒精濃度範圍所沉澱分離的樟芝胞外多醣體，以0.5 mg/ml的劑量處理3T3細胞，反應48小時，移去舊培養基，再以無血清的培養基培養24小時後，檢測3T3細胞分泌MMPs的情形。...............................................................................................67
圖二十二、膠原蛋白濃度檢測之標準曲線...................................................68
圖二十三、50~75% 酒精濃度範圍所沉澱分離的樟芝胞外多醣體，以0.5 mg/ml的劑量處理3T3細胞，反應2、4、8、24、48小時後，檢測培養基內膠原蛋白含量的變化情形。..............................69

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