臺灣博碩士論文加值系統

桑黃(Phellinus linteus)為一眾所皆知寄生於桑樹上的一種橙黃色菇類，大多分布於東方國家。在東方的國家被用作傳統醫學用藥。本研究結果顯示，Phellinus linteus (PL-101)菌種在洋菜培養基探討較適生長條件為溫度30℃；利用木屑太空包可在三個月內將桑黃人工培育出菇；在生物活性之探討中，利用一般常見細菌與真菌做ㄧ抗生活性試驗，且以動物細胞株探討腫瘤細胞存活率、腫瘤細胞轉移試驗、免疫力試驗及抗氧化等功能性分析。
探討抗生活性試驗中，證實了PL-101菌種利用補腦丸藥渣(BN)固體培養所培養之桑黃菌絲體會因萃取溶劑極性不同，而對串珠鐮刀菌(Fusarium moniliforme)的產孢能力具有不同程度抑制效果，其中又以正丁醇萃取物有最顯著的抗菌活性；且十種藥渣固體培養基培養PL-101，對腫瘤細胞存活率也有下降之影響，尤其以桑白皮藥渣(SBB)經桑黃發酵後的熱水萃取物效果最佳，子實體正丁醇萃取物影響五株腫瘤細胞濃度存活率最為顯著，有抑制腫瘤細胞的效果，但對CHO-K1正常細胞其毒性較弱；轉移實驗中顯示經由PL-101發酵後50%EtOH萃取物最有效的降低MMP-9的活性為益母草藥渣(YMETPL)，子實體熱水萃物(FB)及50%EtOH萃取物(FBET)皆可降低MMP-9及MMP-2；免疫力試驗中結果顯示經PL-101菌種培養於桑白皮藥渣(SBB)、補血調經丸藥渣(BX)、六味地黃丸藥渣(LW)、咳嗽固金丸藥渣(CS)及益母草藥渣(YM)固體培養基之熱水萃取液，具有抑制NO產生的能力，50%EtOH萃取部份則以補腦丸藥渣(BNET)較具抑制NO產生的能力，子實體熱水(FB)及50%EtOH萃取物(FBET)皆可降低NO產生的能力；抗氧化實驗中，顯示補腦丸藥渣(BNET)經桑黃發酵後由熱的水及50%EtOH萃取之保護能力最為顯著，子實體熱水(FB)及50%EtOH萃取物(FBET)皆具有保護功能。成分分析方面；將培養於補腦丸藥渣(BN)固體培養基的桑黃菌絲體做初步的薄層分析，因Rf值明顯有差異，初步證實 (PL-101)菌種在補腦丸藥渣(BN)培養基培養前後具明顯差異性。

Abstract
Phellinus linteus, a well-known orange color mushroom and growing on mulberry tree, is scattering around the oriental countries. There has been used as a traditional medicine in oriental countries. The result of this study shows , it is 30 degrees Centigrade of temperature that Phellinus linteus (PL-101 ) fungus plants in the agar culture and probes its righter condition for growing; Utilizing the wood flour can cultivate the PL-101 and artificially the mushroom within three months while Bag log cultivation. In the biological activation, utilize general common bacterium and fungi to make antibiotic test, and using the cell line to test the tumor cell viability, cancer cell metastasis, anti-oxidant, immune assay and the other function.
The mycelial of Phellinus linteus was grown well when it is cultured on the Chinese Herbal Formula Substrates solid media. The growth of mycelial depends on the solid media and the mycelials are all yellow. Penicillium citrinum exhibits the best antibiotic activity when cultured on BN traditional chinese medicine solid media. The ability of inhibition in growing of Penicillium citrinum was affected by the polarity of solvents extracted from Phellinus linteus growing on BN culture.
Phellinus linteus mycelial was grown well when cultured on ten different solid media. The preliminary results indicate the capability of inhibition of tumor cells on the hot water extracts after culturing. Especially in SBB. As the concentration of dose dependence and shoulders with cell's survival rate. But its toxicity is relatively weak in CHO-K1 cell. By the cancer cell metastasis experiment, the most effective reducing MMP-9 of YM Chinese Herbal Formula Substrates(YMETPL)in the PL-101 mycelial 50% Ethanol extracts activation, but the activation of MMP-2 does not have obvious reducing. The result of the immune test shows that the resistibility in inflammation depends on the concentration of hot water extracts of the mycelia cultured on SBB, BX, LW, CS, YM and 50% Ethanol extracts on BNET solid medium. These extracts can inhibit NO activity of mouse macrophage cell (RAW 264.7) stimulated by lipopolysaccharide (LPS 2μg/mL). In the anti-oxidant experiment , show that Chinese Herbal Formula Substrates (BNET ) are most by the protection ability that hot water and 50% Ethanol extracts of to invigorate the BN. The thin layer chromatography illustrates significant difference Rf in composition of Phellinus linteus mycelial before and after culturing.

目次
中文摘要……………………………………………………………………………Ⅰ
英文摘要……………………………………………………………………………Ⅱ
目次…………………………………………………………………………………Ⅲ
表目錄………………………………………………………………………………Ⅴ
圖目錄………………………………………………………………………………Ⅵ
第一章 研究動機……………………………………………………………………1
第二章 文獻回顧……………………………………………………………………2
2.1菇類介紹………………………………………………………………………2
2.2菇類的栽培……………………………………………………………………6
2.3介紹桑黃………………………………………………………………………9
2.3.1桑黃的由來………………………………………………………………9
2.3.2桑黃的分類及特徵……………………………………………………10
2.3.3桑黃的化學成分-游離糖類……………………………………………14
2.3.4桑黃之藥理作用………………………………………………………14
第三章 研究材料與方法……………………………………………………………16
3.1實驗材料……………………………………………………………………16
3.1.1 實驗菌株……………………………………………………………16
3.1.2 科學中藥渣…………………………………………………………16
3.1.3 藥品清單……………………………………………………………16
3.1.4 實驗儀器……………………………………………………………18
3.2實驗方法……………………………………………………………………19
3.2.1 實驗架構……………………………………………………………19
3.2.2 菌種較適生長條件探討活化菌種…………………………………20
3.2.3 培養溫度測試………………………………………………………20
3.2.4 固體培養……………………………………………………………20
3.2.5 樣品備製……………………………………………………………21
3.2.6 抗生物活性試驗……………………………………………………22
3.2.7 細胞存活率試驗……………………………………………………23
3.2.8 轉移試驗……………………………………………………………24
3.2.9 細胞法抗氧化試驗…………………………………………………26
3.2.10 抗發炎試驗…………………………………………………………27
3.2.11成份分析試驗………………………………………………………28
3.2.12 統計分析……………………………………………………………28
第四章 結果與討論………………………………………………………………29
4.1 PL-101菌種較適生長條件之探討………………………………………29
4.1.1 PL-101菌種較適生長溫度…………………………………………29
4.1.2 PL-101菌種較適生長培養基………………………………………30
4.1.3 PL-101菌種實驗室人工栽培太空包………………………………30
4.1.4 十種固體培養基與PL-101菌種熱水及50%EtOH萃取物之產率…34
4.1.5 十種固體培養基與PL-101菌種熱水萃取物之pH值………………35
4.1.6 補腦丸藥渣固體培養基與PL-101菌種溶劑萃取物之pH值……36
4.1.7 十種藥渣與經PL-101菌種發酵之50%EtOH萃取物相對酒精含量…37
4.2 抗生物活性之探討…………………………………………………………39
4.3 MTT 試驗…………………………………………………………………42
4.4 轉移試驗…………………………………………………………………68
4.5 細胞法抗氧化試驗………………………………………………………75
4.6 抗發炎試驗………………………………………………………………83
4.7 成份分析試驗……………………………………………………………91
第五章 總結………………………………………………………………………93
參考文獻……………………………………………………………………………95
表目錄
表1 多種菇類的組成成分與其抗腫瘤活性 ………………………………………5
表2 十種固體培養基與PL-101菌種熱水及50%EtOH萃取物之產率…………34
表3 十種固體培養基與PL-101菌種熱水萃取影響之pH值……………………35
表4 補腦丸藥渣(BN)固體培養基與PL-101菌種影響溶劑萃取物之pH值……36
表5 十種藥渣與經PL-101菌種發酵之50%EtOH萃取物相對酒精含量………38
表6 固態栽培PL-101菌種菌絲體熱水萃取物之抗菌能力………………………40
圖目錄
圖一 菇類的子實體及菌絲體部位名稱圖…………………………………………8
圖二 桑黃的分類……………………………………………………………………11
圖三 段木栽培之桑黃子實體………………………………………………………12
圖四 培養於平板培養皿上的桑黃菌絲體…………………………………………12
圖五 顯微鏡下桑黃菌絲體…………………………………………………………13
圖六 溫度對PL-101菌絲生長速率之影響…………………………………………29
圖七 固體培養基所培養之PL-101菌絲體…………………………………………31
圖八 固體培養基所培養之PL-101菌絲體 ………………………………………32
圖九 桑黃子實體……………………………………………………………………33
圖十 濃度為2.5%及5%的EtOH對腫瘤細胞存活能力之影響………………38
圖十一 補腦丸藥渣及經PL-101菌種對串珠鐮刀菌產孢能力…………………41
圖十二 藥渣及經PL-101發酵熱水萃取物對子宮頸癌細胞存活能力之影響…43
圖十三 藥渣及經PL-101發酵熱水萃取物對纖維母細胞存活能力之影響…44
圖十四 藥渣及經PL-101發酵熱水萃取物對前列腺癌細胞存活能力之影響45
圖十五 藥渣及經PL-101發酵熱水萃取物對乳癌細胞存活能力之影響…46
圖十六 藥渣及經PL-101發酵熱水萃取物對大腸癌細胞存活能力之影響…47
圖十七 藥渣及經PL-101發酵酒精萃取物對子宮頸癌細胞存活能力之影響…49
圖十八 藥渣及經PL-101發酵酒精萃取物對纖維母細胞存活能力之影響…50
圖十九 藥渣及經PL-101發酵酒精萃取物對前列腺癌細胞存活能力之影響…51
圖二十 藥渣及經PL-101發酵酒精萃取物對乳癌細胞存活能力之影響…52
圖二十一 藥渣及經PL-101發酵酒精萃取物對大腸癌細胞存活能力之影響…53
圖二十二 SBB及其經PL-101發酵熱水萃取物對腫瘤細胞存活率……55
圖二十三 BX及其經PL-101發酵熱水萃取物對腫瘤細胞存活率……56
圖二十四 LW及其經PL-101發酵熱水萃取物對腫瘤細胞存活率……57
圖二十五 BN及其經PL-101發酵熱水萃取物對腫瘤細胞存活率……58
圖二十六 SBB及其經PL-101發酵酒精萃取物對腫瘤細胞存活率……60
圖二十七 BX及其經PL-101發酵酒精萃取物對腫瘤細胞存活率……61
圖二十八 BN及其經PL-101發酵酒精萃取物對腫瘤細胞存活率……62
圖二十九 GD及其經PL-101發酵酒精萃取物對腫瘤細胞存活率……63
圖三十 桑黃子實體溶劑萃取物降低腫瘤細胞存活率…………………………64
圖三十一 藥渣及經PL-101發酵熱水萃取物對正常細胞之存活能力………66
圖三十二 桑黃子實體溶劑萃取物對正常細胞之存活能力………………………67
圖三十三 由PMA所誘導之MMP-2及MMP-9活性………………………………69
圖三十四 由PMA所誘導之MMP-2及MMP-9活性……………………………70
圖三十五 由PMA所誘導之MMP-2及MMP-9活性………………………………71
圖三十六 由PMA所誘導之MMP-2及MMP-9活性………………………………72
圖三十七 由PMA所誘導之MMP-2及MMP-9活性………………………………73
圖三十八 由PMA所誘導之MMP-2及MMP-9活性………………………………74
圖三十九 H2O2(1.8mM)對HeLa細胞存活之半抑制濃……………………………76
圖四十 細胞法抗氧化活性………………………………………………………77
圖四十一 細胞法抗氧化活性………………………………………………………78
圖四十二 細胞法抗氧化活性………………………………………………………79
圖四十三 細胞法抗氧化活性………………………………………………………80
圖四十四 細胞法抗氧化活性………………………………………………………81
圖四十五 細胞法抗氧化活性………………………………………………………82
圖四十六 Nitrite assay檢量線………………………………………………………84
圖四十七 藥渣及經PL-101發酵熱水萃取液之抗發炎測試………………………85
圖四十八 藥渣及經PL-101發酵酒精萃取液之抗發炎測試………………………86
圖四十九 SBB及BY經PL-101發酵熱水萃取液之抗發炎測試…………………87
圖五十 YM及LW經PL-101發酵熱水萃取液之抗發炎測……………………88
圖五十一 CS及BN經PL-101發酵熱水及酒精萃取液之抗發炎測………………89
圖五十二 桑黃子實體熱水及酒精萃取之抗發炎測試……………………………90
圖五十三 BN及經PL-101發酵熱水萃取物之薄層分析…………………………86

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