臺灣博碩士論文加值系統

本研究將樟芝深層培養液(ACSC)分為菌絲體和發酵濾液的多醣體，其中又將菌絲體分為以冷水萃取及熱水萃取兩種多醣體，再將上述三種多醣體(PEF、PEMC、PEMH)予以透析處理(cut off 12 KD)，取得分子量較大的部分(h-PEF、h-PEMC、h-PEMH)。取上述六種多醣體與發酵濾液乾燥物(DMF)及菌絲體水萃取物(WEM)進行抗氧化力評估，以了解多醣體是否為發酵濾液及菌絲體水萃取物之主要抗氧化物。此外，評估六種多醣體在肝細胞模式系統(Chang liver cells)之抗氧化能力，並探討其在胞內具有抗氧化能力的可能機制。最後進行高分子量多醣體(MW>12 KD)之分離純化及其單糖組成分析。
研究中先以氧自由基吸收能力(oxygen radical absorbance capacity assay; ORAC)來評估DMF、WEM及六種不同多醣體的抗氧化能力，結果顯示六種多醣體中，以菌絲體熱水萃取多醣體(PEMH及h-PEMH)之抗氧化效果最佳(約3.4 M Trolox equivalent)。
胞內系統之抗氧化力評估乃是以彗星試驗法(comet assay)及測定DNA oxidative adduct (8-hydroxydeoxyguanosine; 8-OHdG)的含量來評估六種多醣體對H2O2誘導DNA氧化傷害之保護及修補能力。實驗設計分為兩種，一種是先將六種多醣體與Chang liver cells預培養5小時後，再加入H2O2 (50 M, 30 min)誘導細胞氧化傷害；另一種則是先加入H2O2 (50 M, 30 min)誘導細胞氧化傷害後，再加入多醣體與Chang liver cells培養5小時，其目的乃為探討多醣體保護及修補DNA的能力。以彗星試驗法評估六種多醣體對細胞DNA保護作用的結果顯示，以PEMH及h-PEMH的保護效果最顯著，在200 g/ml之濃度下，% tail DNA從43%下降至8%，約抑制81%之DNA氧化傷害；另外，六種多醣體亦均能抑制H2O2所誘導8-OHdG氧化產物的產生，經透析後之多醣體其抑制率約有50-60%，而粗多醣體則可完全抑制8-OHdG氧化產物的產生。由彗星試驗法評估六種多醣體對細胞DNA修補作用的結果可知，六種多醣體均具有修補DNA損傷的能力，在200 g/ml之濃度下，% tail DNA從41%下降至15-25%，約抑制46-74%之DNA氧化傷害；且六種多醣體亦均能完全修補H2O2所誘導8-OHdG氧化產物的生成。
進一步再以人類肝細胞(Chang liver cells)為模式系統，探討加入ACSC之多醣體，對於胞內各種抗氧化酵素活性之影響。結果顯示六種多醣體對glutathione S-transferase (GST)酵素活性均有增加之趨勢，其中h-PEMC、PEMH及PEF在與細胞預反應後，均可使酵素活性提升至原來酵素活性之1.49、1.62及1.50倍。但相對地卻使胞內之glutathione peroxidase (GPx)及glutathione reductase (GRd)之酵素活性下降，其中尤以h-PEMH、h-PEF及PEMH下降率最大，其酵素活性GPx及GRd之下降率分別為66~71 %及83~89 %。然在GSH/GSSG ratio的測定上，其ratio都是上升，因此顯示其具有提昇胞內抗氧化防禦狀態之能力。
將ACSC之多醣體純化，並進行其單糖組成之分析。結果在純化過程中h-PEMH僅得到一個吸收波峰，且與蛋白質鍵結；h-PEMC和h-PEF均有兩個吸收波峰，但h-PEMC得到一個蛋白質鍵結的多醣體(h-PEMC-1)及一個未與蛋白質結合的多醣體(h-PEMC-2)，而h-PEF的兩個吸收波峰則均未鍵結蛋白質。之後並測得這六種ACSC多醣體的單糖組成主要成分是葡萄糖，另外尚有木糖、半乳糖、阿拉伯糖、山梨糖等單糖。
綜合上述結果推論ACSC之多醣體可藉由提昇GST酵素活性之能力，來提昇H2O2之代謝能力，因此可抑制脂質過氧化作用，並且維持GSH/GSSG ratio在還原狀態下，減少DNA damage以及細胞毒性，由此而保護DNA避免氧化傷害。

The aim of this thesis was to investigate the antioxidant properties of six polysaccharides that extracted from Antrodia camphorata in submerged culture (ACSC) using Chang liver cells. Polysaccharide extracts were prepared from filtrate (PEF) and mycelia of ACSC. The mycelia of ACSC were extracted with cold and hot water to obtain the polysaccharide PEMC and PEMH, respectively. PEMC, PEMH and PEF were dialyzed (MW cut off =12 KD) to obtain large MW polysaccharides (h-PEMC, h-PEMH and h-PEF). To estimate antioxidant properties of those polysaccharides, oxygen radical absorbance capacity (ORAC) assay was used. To determine DNA protective effects on H2O2-treated (50 M) Chang liver cells, six polysaccharides were pre-incubated with cells for 5h and then treated with H2O2 for the further 30 min. On the other hand, a reverse treatment order of polysaccharides and H2O2, as mention above, were also employed in DNA repair study. DNA damage was monitored with comet assay and 8-hydroxydeoxy-guanosine (8-OHdG) formation. As to antioxidant mechanisms confirming, we determined the antioxidant defense system (glutathione peroxidase, GPx; glutathione reductase, GRd; Glutathione S-transferase, GST; and both reduced and oxidized form of glutathione, GSH & GSSG) and intracellular reactive oxygen species (ROS) levels in polysaccharides per-incubated Chang liver cells (ultimately treat with H2O2).
PEMH and h-PEMH was the top two polysaccharides with an ORAC value about 3.4 M trolox equivalent. In addition to antioxidant property, PEMH and h-PEMH (200 g/ml) pre-incubated with Chang liver cells could reduce the DNA damage induced by following H2O2 treatment (% tail DNA reduced from 43 to 8%). In the same experimental conditions, the 8-OHdG content was diminished than control in Chang liver cells that had been pre-incubated with all the six polysaccharides. In the DNA repair study, all the six polysaccharides repaired DNA damage (% tail DNA reduced from 41% to a range between 15-25%) and reduced 8-OHdG content completely in H2O2-treated Chang liver cells. These results indicated that polysaccharides extract from ACSC might protect and repair DNA damage in oxidative treat cells (depend on treatment orders).
When Chang liver cells pre-incubated with those polysaccharides and H2O2 in order, the GST activity in h-PEMC, PEMH and PEF were increased (1.62, 1.50, and 1.49 times, respectively). On the contrary, GPx and GRd activity were decreased. The GSH/GSSG ratio, however, raised under the same experimental condition. This result revealed that the protective and reparative effects of polysaccharides extracted from ACSC might be associated with GST activity increasing and the Chang of intracellular antioxidant status. Finally, we purified and analyzed the sugar constituents of high MW polysaccharides. In the whole chromatographic condition, h-PEMH showed only one peak and it was a polysaccharides-protein complex; h-PEMC showed two peaks and one of which bound with protein (h-PEMC-1), but the other was not (h-PEMC-2); and the last h-PEF showed two peaks which were all pure polysaccharides. The main sugar component of the six polysaccharides was glucose, and moreover, xylose, galactose, arabinose, and mannose were the else.
In conclusion, polysaccharides extracts from ASCS is able to protect cell oxidative damage of cell induced by H2O2. The possible mechanisms may involve in GST activity raising, intracellular antioxidant status altering.

目 錄
中文摘要…………………………………………………………………Ⅰ
英文摘要…………………………………………………………………Ⅲ
前言………………………………………………………………………1
文獻整理…………………………………………………………………3
一、 菇類簡介………………………………………………………3
1. 菇類的營養特性(一次機能性) ………………………………3
2. 菇類的嗜好特性(二次機能性)………………………………5
3. 菇類的生理特性(三次機能性)………………………………6
a. 抗腫瘤物質……………………………………………………7
b. 降血壓物質………………………………………………………9
c. 降血糖物質………………………………………………………9
d. 降低膽固醇物質…………………………………………………9
e. 抗炎症物質………………………………………………………9
二、氧化壓力對人體的影響……………………………………………10
(一)、氧化壓力對細胞所造成之影響……………………………11
(二)、體內的抗氧化系統…………………………………………12
三、菇類之相關研究報告…………………………………………….15
1. 生理活性之相關研究……………………………………………15
2. 活性成分-1,3-D-glucan之結構分析………………………20
四、菇類多醣體之機能性研究………………………………………23
1. 結構分析………………………………………………………23
2. 多醣體之簡介……………………………………………………27
3. 多醣體調節人體生理活性之功能………………………………27
五、菇類其他機能性成分之研究………………………………………29
六、樟芝簡介及其功能性研究…………………………………………30
七、研究目的……………………………………………………………34
實驗流程………………………………………………………………………35
材料與方法……………………………………………………………………36
結果與討論……………………………………………………………………50
一、 濾液及菌絲體之多醣體萃取率及其含量分析…………………50
二、抗氧化力之測定--氧自由基吸收能力之測定 (Oxygen radical absorbance capacity assay; ORAC)……………………………………52
三、對chang liver cells 存活率之影響………………………………52
1. 樟芝深層培養液 (ACSC)之多醣體的MTT測試………………52
2. 樟芝深層培養液(ACSC)之多醣體對DNA之影響………………54
A. 彗星試驗法……………………………………………………54
B. 8-OHdG 分析……………………………………………………57
四、以彗星試驗法評估ACSC多醣體對H2O2誘發人類肝細胞(Chang liver cells) DNA損傷之影響……………………………………………………57
1. Cells + polysaccharide extracts + H2O2………………………57
2. Cells + H2O2 + polysaccharide extracts………………………60
五、以8-OHdG生成量分析ACSC多醣體對H2O2誘導人類肝細胞(Chang liver cells) DNA傷害之影響……………………………………………60
1. Cells + polysaccharide extracts + H2O2………………………60
2. Cells + H2O2 + polysaccharide extracts………………………63
六、樟芝深層培養液之多醣體對chang liver cells胞內活性氧(ROS)生成之影響………………………………………………………………………63
七、ACSC多醣體抑制胞內脂質過氧化物(MDA)形成之能力………………65
八、 樟芝深層培養液多醣體對胞內抗氧化酵素活性之影響………68
1. GST酵素活性……………………………………………………68
2.GPx酵素活性…………………………………………………………69
3. GRd酵素活性…………………………………………………………69
九、 GSH/GSSG ratio之測定………………………………………………72
十、樟芝深層培養液(ACSC)之濾液及菌絲體多醣萃取物之區分………75
十一、 透析前、後樟芝深層培養液之濾液及多醣體的萃取率、蛋白質及多醣體糖組成之分析………………………………………………………77
結論……………………………………………………………………………83
參考文獻………………………………………………………………………85

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