臺灣博碩士論文加值系統

樟芝（Antrodia cinnamomea）是台灣特有食藥用真菌，近年來已有多份研究報告指出樟芝具有提升免疫機能、抑制腫瘤細胞生長等功效。由於其子實體之生長緩慢，且栽培不易，應用液態大量培養菌絲體，是目前生技產業普遍採用之方式。菌絲體中以多醣體應用於炎症相關的疾病預防為主，在癌症治療及預防上亦備受關注。本研究藉由樟芝菌絲體存在豐富的多醣體中，製備並純化具活性之多醣體，探討其結構與組成，並分別應用至細胞與動物之研究，了解其抗發炎之功效。
樟芝菌絲體首先利用熱水萃取去除可溶性多醣後，繼續以鹼萃取再利用等電點沉澱方式而獲得鹼可溶粗多醣，再以Sepharose CL-6B樹脂進行膠體層析以純化多醣，將純化後的多醣體(AC-IIb)進行總醣、總蛋白、醛糖酸、-葡聚醣、單糖與胺基酸組成分析、分子量測定、紅外線光譜分析與核磁共振光譜分析。進一步以小鼠巨噬細胞RAW264.7進行AC-IIb之生物活性測試，探討AC-IIb預防脂多醣(lipopolysaccaride, LPS)誘發RAW 264.7細胞發炎之機制，以及透過大鼠之動物實驗確認AC-IIb在體內中抗發炎之效果。
結果顯示，菌絲體中多醣體AC-IIb之收率為 9.19 % (w/w, dw)，其中主要為蛋白質(71%)之組成，醣類佔14.1%，屬於醣蛋白之結構，蛋白質中以leucine含量比例最高(18.49%)，其次是valine (13.76%)，單糖類中則以xylose為主(53%)，其次是glucose (21%)，因此純化之多醣體係屬於醣蛋白(glycoprotein)之一種，分子量為440 kDa 多醣結構中-葡聚醣佔14.20%，其鍵結結構包括-1,3- 與-1,4-糖苷鍵。
在保護巨噬細胞免於脂多醣誘發之發炎反應中，發現當AC-IIb處理24、48小時及濃度在18.75 g/ml以上時，有顯著的抑制脂多醣誘發一氧化氮生成之能力，而在72小時及濃度12.5 g/ml以上時，即有顯著抑制脂多醣誘發一氧化氮生成之能力，此結果顯示AC-IIb在脂多醣誘導巨噬細胞中具有抗發炎之效果，進一步以大鼠進行試驗，探討AC-IIb於體內活性表現之程度。
實驗以胃管灌食方式進行，探討AC-IIb於脂多醣所誘發雄性大鼠(Sprague-Dawley)急性肝損傷之保護能力，實驗共分五組，包括控制組(control)、樟芝醣蛋白組(AC-IIb, 40 mg/kg)、脂多醣組(lipopolysaccaride, LPS)、脂多醣暨樟芝醣蛋白組(低劑量組AC-IIb L: 40 mg/kg + LPS 與高劑量組AC-IIb H: 80 mg/kg + LPS)，實驗為期一周，於第七天以腹腔注射方式施打脂多醣 (5 mg/kg B.W.)，24小時後犧牲實驗動物進行分析，觀察大鼠血清中麩草酸轉氨基酶(GOT)、麩丙酮轉胺基酶(GPT)、一氧化氮(NO)及介白素-6(IL-6)之含量變化，以及肝臟組織中丙二醛(MDA)與抗氧化酵素之含量(SOD、CAT、GSH-Px)，此外，以西方墨點法分析肝臟蛋白質中，相關發炎蛋白之表現程度。實驗結果發現，當與控制組相比，脂多醣組的發炎反應因子濃度皆顯著增加，一氧化氮濃度增加7.7倍、介白素-6濃度增加2.9倍、肝臟中脂質過氧化產物含量增加1.4倍，而抗氧化酵素之活性皆為下降 (SOD下降 2.8倍、CAT下降3.4倍、GSH-Px下降3.8倍)，顯示經脂多醣處理會造成組織的傷害。在給予不同濃度之樟芝醣蛋白組(AC-IIb L and AC-IIb H)前處理，對脂多醣的傷害都有明顯的保護作用。在AC-IIb L組與AC-IIb H比LPS組GOT含量下降了1.5倍與1.6倍，在GPT含量下降了5.6倍與4.3倍，一氧化氮含量下降了5.3倍與3.9倍，介白素-6含量下降了2倍與1.3倍，脂質過氧化下降了1.2倍與1.8倍，在抗氧化酵素活性含量(SOD上升1.5倍與1.9倍、CAT上升了1.3倍與1倍、GSH-Px上升了1.3倍與0.9倍)；此外，藉由肝組織內與發炎相關之蛋白質表現分析，包括NF-B, i-NOS等，顯示多醣體AC-IIb具有減緩脂多醣誘發之自由基與發炎因子對肝臟組織功能之影響。

The fruiting body of Antrodia cinnamomea, a rare and expensive medicinal fungus, is exclusively grown on the rotten wood Cinnamomun kaehirae in Taiwan. There have been many biological activities reported from the literatures, including immune modularity and tumor growth inhibition. Owing to the limited resources in nature, the submerged liquid fermentation of A. camphorata for producing the mycelium has been developed by local biotechnology industry. However, the polysaccharides are the major composition existed in the mycelium different from that of fruiting body with triterpenoids as the important composition. In recent years, the mycelium has been mostly used for the prevention of inflammation-related diseases. However, there is no report of anti-inflammatory effects of the purified polysaccharide or its biological mechanism from the fermented mycelial. In this study, we firstly purified and characterized the chemical composition of the mycelium of A. cinnamomea, and then investigated the anti-inflammatory effects of the polysaccharide and its biological mechanism in lipopolysaccharide (LPS)-induced inflammatory responses in macrophages and animals.
In the purification of polysaccharide, the soluble polysaccharides were removed from the mycelia of A. cinnamomea by using hot water before proceeding to purify the obtained polysaccharides. The alkaline extraction and acid precipitation were then used to isolate the polysaccharides for further purification by using gel filtration chromatography with Sepharose CL-6B to obtain the purified polysaccharide (AC-IIb). The chemical characterization including total sugar, total protein, uronic acid, β-glucan, monosaccharides and amino acids composition, molecular weight determination, FTIR and NMR analyses were investigated to determine the molecular structure of AC-IIb. Results showed that protein (71.0%) is the major composition higher than that of, carbohydrate (14.1% ) in AC-IIb being characterized as a glycoprotein. In amino acids composition, leucine (18.49%) was the highest content and valine (13.76%) was the second. In monosaccaride composition, xylose (53%) and glucose (21%) were the majors. The molecular structure of AC-IIb was determined as -1,3- and -1,4-linkages with the average molecular weight of 440 kDa.
In the prevention effect of AC-IIb from the lipopolysaccharide induced inflammatory responses on macrophages RAW 264.7, AC-IIb treatment (18.75 g/ml) could significantly reduced the nitrogen oxide production at 24 and 48 hours. While in the treatment of AC-IIb (12.5 g/ml) at 72 hours, the significant reduction of nitrogen oxide was showed in the LPS-induced macrophages.
This study suggests that AC-IIb exerts anti-inflammatory effects by down-regulating the production and expression of pro-inflammatory cytokines and mediators via inhibiting the NF-κB pathway in LPS-stimulated RAW 264.7 cells. The in vivo study was then forwarded to investigate the anti-inflammatory bioactivities of purified polysaccharide AC-IIb
Animal study were investigated with 7-day of short-term feeding experiments with AC-IIb on LPS-induced liver damage in male Sprague-Dawley rats. The studies were divided into five groups, including control group, AC-IIb group (40 mg/kg b.w.), LPS group (5 mg/kg b.w.), AC-IIb (40 mg/kg and 80 mg/kg b.w.) plus LPS groups for one week. LPS (5 mg/kg b.w.) was introduced intraperitoneally at the 7th day and animals were sacrificed 24 h post-LPS challenge for analysis. In hepatic damages analyses, serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities were significantly up-regulated by LPS treatment. The inflammatory factors of NO and IL-6 in serum and the levels of lipid peroxidation product (malondialdehyde) and the activities of antioxidant enzymes (SOD, CAT and GSH-Px) in the liver tissue, were also significant induced after the LPS treatment as evidenced by the activation of inflammatory responses (NO and IL-6 increased 7.7 and 2.9 fold, respectively); increasing in the level of lipid peroxidation product (hepatic MDA increased for 1.4 fold); decreasing in the activities of antioxidant enzymes (liver tissue SOD activities reduced for 2.8 fold, CAT activities reduced for 3.4 fold and GSH-Px activities reduced for 3.8 fold), as compared with control group, indicating that LPS could induce the acute inflammatory responses. Pre-treating the animals with different concentrations of AC-IIb (40 and 80 mg/Kg) before the challenges of LPS resulted in a significant protection against LPS-induced damage, AC-IIb prevention could reduce the LPS-induced tissue injuries (GOT content decreased 1.5 and 1.6 fold, GPT reduced 5.6 and 4.3 fold, NO decreased 5.3 and 3.9 fold, IL-6 decreased 2 and 1.3 fold, MDA decreased 1.2 and 1.8 fold, respectively in low, 40 and high, 80 mg/Kg dosages), and increase levels of antioxidant enzymes (SOD, 1.5 and 1.9 fold, CAT, 1.3 and 1 fold and GSH-Px, 1.3 and 0.9 fold, respectively in low and high dosages). In addition, the protein expressions of inflammatory relatives through the Western blot analysis, including i-NOS and NF-B, indicated that the AC-IIb prepared from A. cinnamomea mycelia could activate the antioxidant enzymes activities in liver tissue and reduce the inflammatory cytokines in serum for attenuating the LPS damages.

目錄.......................................................................................................I
圖目錄..................................................................................................V
表目錄..............................................................................................VIII
中文摘要.............................................................................................IX
英文摘要.............................................................................................XI
第一章 緒論……………………………………...……………………..1
第二章 文獻整理.............................................................................2
第一節、 樟芝(Antrodia cinnamomea)介紹……………….….……2
第二節、 多醣體(polysaccharide)……………………………...……..9
第三節、 -(1,3)-D-葡聚醣………………………..…….…………16
第四節、 醣蛋白(glycoprotein）…………………….…………......20
第五節、 醣類分析方法……………………………………………22
第六節、 巨噬細胞與免疫反應……………………………...……25
第七節、 抗氧化防禦系統…………………………………………31
第三章 實驗架構…………….........................................................34
第四章 材料與方法........................................................................35
第一節 實驗材料與試劑................................................................35
第二節 多醣體萃取製備................................................................38
第三節 多醣化學分析....................................................................40
第四節 細胞培養...........................................................................46
第五節 樟芝多醣AC-IIb抗發炎動物模式試驗..............................48
第六節 實驗動物血液生化值之測定.............................................49
第七節 一氧化氮(NO)濃度之分析................................................49
第八節 酵素免液分析法................................................................49
第九節 脂質過氧化之分析............................................................50
第十節 抗氧化酵素活性之測定.....................................................50
第十一節 西方墨點法.................................................................54
第十二節 組織切片.....................................................................56
第十三節 統計分析.....................................................................56
第五章 實驗結果.........................................................................57
第一節 菌絲體與市售產品Bio bran多醣體萃取率........................57
第二節 以Sephose CL-6B管柱純化樟芝多醣(AC-II)之層析圖.....58
第三節 純化之多醣體AC-IIb總醣與總蛋白之定量分析.............59
第四節 多醣體AC-IIb中醛醣酸之分析.......................................60
第五節 多醣體AC-IIb 單糖組成之氣相層析質譜分析...............61
第六節 多醣體AC-IIb之胺基酸組成分析....................................63
第七節 多醣體AC-IIb分子量之測定...........................................65
第八節 多醣體AC-IIb之-葡聚醣定量分析……………………...69
第九節 多醣體AC-IIb傅立葉轉換紅外線光譜分析….................71
第十節 多醣體AC-IIb核磁共振光譜分析....................................72
第十一節 脂多醣誘發RAW264.7細胞生成一氧化氮(NO)之分
析……………………………………………………......74
第十二節 DMSO對RAW264.7細胞生長影響之分析………..….75
第十三節 多醣體AC-IIb及Bio bran對RAW264.7細胞生長影響
之分析……………………………………………….....76
第十四節 多醣體AC-IIb及市售產品Bio bran多醣體抑制脂多醣
誘導RAW264.7細胞產生一氧化氮…………...……..77
第十五節 大鼠餵食多醣體AC-IIb七天後體重與器官組織重量之
變化….......................................................................80
第十六節 多醣體AC-IIb減緩脂多醣誘發大鼠血清中麩草酸轉胺
基酶(GOT)及麩胺酸丙酮酸轉胺酶(GPT)的上升.......81
第十七節 多醣體AC-IIb抑制脂多醣誘發大鼠血清中一氧化氮
(NO)之生成...............................................................83
第十八節 多醣體AC-IIb抑制脂多醣誘發大鼠血清中介白素-6
(IL-6)之生成..............................................................85
第十九節 多醣體AC-IIb抑制脂多醣誘發大鼠肝臟組織中
丙二醛(MDA)之生成...................................................87
第二十節 多醣體AC-IIb對脂多醣誘導大鼠肝臟組織中超氧岐化
酶(SOD)之影響.........................................................89
第二十一節 多醣體AC-IIb對脂多醣誘導大鼠肝臟組織中過氧化氫
酶(CAT)之影響..........................................................91
第二十二節 多醣體AC-IIb對脂多醣誘導大鼠肝臟組織中麩胱苷肽
過氧化酶(GSH-Px)之影響..........................................93
第二十三節 多醣體AC-IIb對脂多醣誘導大鼠肝臟中一氧化氮合成
酶(iNOS)及核轉錄因子(NF-kB)的蛋白質表現之影響.95
第二十四節 多醣體AC-IIb抑制脂多醣誘發大鼠肝臟組織型
態之變化…………………………………......................97
第六章 討論...................................................................................99
第七章 結論.................................................................................103
第八章 參考文獻.........................................................................104



圖 目 錄
圖一、以Sepharose CL-6B管柱分離樟芝多醣(AC-IIb)之層析圖.....58
圖二、氣相層析質譜儀分析AC-IIb中單糖組成之總離子層析圖....61
圖三、多醣體標準品pullulan分子量之線性回歸曲線..................65
圖四、多醣體之液相層析圖譜.........................................................66
圖五、多醣體AC-IIb之傅立葉轉換紅外光譜…............................71
圖六、多醣體之核磁共振光譜圖…………………….........................72
圖七、脂多醣對巨噬細胞RAW264.7生長之影響.......................73
圖八、脂多醣誘發巨噬細胞RAW264.7一氧化氮之生成….….....74
圖九、DMSO對RAW264.7 細胞生長之影響..............................75
圖十、多醣體AC-IIb及市售產品Bio bran多醣體對RAW264.7細胞生
長影響之分..…….....................................................................76
圖十一、多醣體AC-IIb與市售產品Bio bran多醣體對脂多醣誘發
RAW264.7細胞一氧化氮生成之影響...................................77
圖十二、多醣體AC-IIb減緩脂多醣誘發大鼠血清中麩草酸轉胺
基酶(GOT)及麩胺酸丙酮酸轉胺酶(GPT)活性的上升.......82
圖十三、多醣體AC-IIb抑制脂多醣誘導大鼠血清中亞硝酸鹽
(Nitrite)之生成................................................................84
圖十四、多醣體AC-IIb抑制脂多醣誘導大鼠血清中介白素-6(IL-6)
之生成..............................................................................86
圖十五、多醣體AC-IIb抑制脂多醣誘導大鼠肝臟組織中丙二醛
(MDA)之生成.……………….…….……………………….88
圖十六、多醣體AC-IIb減緩脂多醣誘發導大鼠肝臟組織中超氧岐化酶
(SOD)活性的改變………………………………………….....90
圖十七、多醣體AC-IIb減緩脂多醣誘導大鼠肝臟組織中過氧化氫酶
(CAT) 活性的改變................................................................92
圖十八、多醣體AC-IIb對脂多醣誘導大鼠肝臟組織中麩胱苷肽過氧
化氫酶(GSH-Px)活性的改變...............................................94
圖十九、多醣體AC-IIb對脂多醣誘導大鼠肝臟組織中誘導型一氧化氮
合成酶(iNOS)及核轉錄因子(NF-kB)蛋白質表現量之影響...96
圖二十、多醣體AC-IIb改善脂多醣誘導大鼠組織型態學的變化….....98

表 目 錄
表一、 樟芝生理活性論文之整理........................................................7
表二、 列出各種不同真菌之多糖的種類、來源其生理功能…...........12
表三、純化之多醣體AC-IIb總糖及總蛋白之含量…………………..59
表四、多醣體AC-IIb之醣醛酸含量…………………………….60
表五、多醣體AC-IIb之單糖組成……………………………..………..62
表六、多醣體AC-IIb之胺基酸組成分析….………………….………..63
表七、多醣體AC-IIb與Bio bran多醣體之平均分子量…….………..68
表八、多醣體AC-IIb之與-葡聚醣含量……………………….....69
表九、餵食多醣體AC-IIb對大鼠體重變化與組織重量變化之比值..80



附 圖 目 錄
附圖一、固態段木栽培牛樟芝...............................................................4
附圖二、樟芝菌絲體的態.......................................................................4
附圖三、牛樟樹之型態..........................................................................5
附圖四、多醣體破壞病源體激活免疫細胞的示意圖...........................10
附圖五、-glucan對人體免疫與癌細胞之影響…………………………14
附圖六、具抗腫瘤活性的-(1→6)分枝與-(1→3)-D-葡聚醣結...........15
附圖七、具抗腫瘤活性-(1→6)分枝與-(1→3)-D-葡聚醣三股螺旋結
晶構型............................................................................15
附圖八、-葡聚醣在巨噬細胞中之訊息傳遞路徑. .............................17
附圖九、-（1→3）-D-葡聚醣的三重螺旋結構示意圖..………….........19
附圖十、多醣體/蛋白質複合物的分子結構示意圖..............................20
附圖十一、醣蛋白中多醣與蛋白質鍵結方式.......................................21
附圖十二、脂多醣的化學結構.............................................................26
附圖十三、一氧化氮合成途徑.............................................................27
附圖十四、IL-6於急性及慢性發炎反應中扮演的角色........................30

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