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研究生:林雪娥
研究生(外文):Hsueh-O Lin
論文名稱:血紅密孔菌(Pycnoporussanguineus)栽植體之成份分析與抗發炎活性研究
論文名稱(外文):Studies on chemical constituents and anti-inflammatory activity of cultivated Pycnoporus sanguineus
指導教授:徐鳳麟
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:143
中文關鍵詞:血紅密孔菌
外文關鍵詞:Pycnoporus sanguineus
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血紅密孔菌 (Pycnoporus sanguineus (Fr.) Murr) 泛佈於熱帶和亞熱帶地區,為一種木材腐朽菌,野生子實體生長速度較為緩慢,因此造成研究材料之不足,本研究針對培養之菌絲體進行生成之生物量及化學成分研究。結果顯示血紅密孔菌在培養49 天後,菌絲乾重達最高產率16.13±2.10 g/l,而多醣體產量在培養 21 天時,達1.02±0.40 g/l最高。培養第28 天時乙醇萃取物產量為6.10±1.90 g/l 最高。而所含多醣體之單醣組成分析,結果顯示以岩藻醣 (fucose)、葡萄糖 (glucose)及甘露糖 (mannose) 為主要單糖組成,其含量分別為64.81±0.15,18.70±0.05, 17.56±0.03 μmol/g PS。針對血紅密孔菌菌絲體進行分離純化,共得到四個麥角甾醇,分別為:ergosta-7,22-dien-3β-ol (ST-1: 1),3β,5α-dihydroxyergosta-7,22-dien-6-one (ST-2: 2), 3β,5α,9α-trihydroxy-ergosta-7,22-dien-6-one (ST-3: 3)及3β,5α,6α-trihydroxyergosta-7,22-diene (ST-4: 4)。ST-2, ST-3, ST-4 三個化合物是首次自血紅密孔菌中分離得到之已知化合物。藥理活性測試以LPS 誘導老鼠微神經膠細胞株 (BV-2) 產生NO 的抗發炎模式篩檢中,乙醇粗萃物在50 μg/ml濃度下,抑制率達87.5 %,具有極顯著抑制NO 產生的效果。乙醇粗萃物之劃分,乙酸乙酯層在20 μg/ml 濃度下,抑制率達87.5 %,正丁醇層20 μg/ml 濃度時,抑制率45.5 %。經單離後之純化合物ST-1對LPS 誘導之微神經膠細胞株在15 μM 濃度具有43.5 % 的抑制率。
Pycnoporus sanguineus (Fr.) Murr is a slow-growing saprophytic fungus which causes decay of certain types of wood in the forests of tropical and subtropical areas. Because of the material is limited on the island and poor growth rate in nature, liguid culture of the fungus was
attempted. The results showed that the fungus was cultured for 49 days to reach the maximal dry weight of 16.13±2.10 g/l. The yield of polysaccharides (PS) of P. sanguineus for 21 days was 1.02±0.40 g/l. The yield of ethanolic extract of mycelial of P. sanguineus for 28 days was
6.10±1.90 g/l. Compositional analysis PS of P. sanguineus showed that fucose, glucose and mannose were the predominant sugars in the value of 64.81±0.15, 18.70±0.05 and 17.56±0.03 μmol/g PS, respectively.
Bioassay-guided fractionation was led to the isolation of four ergostane skeleton type steroids, including ergosta-7,22-dien-3β-ol (ST-1: 1), 3β,5α-dihydroxyergosta-7,22-dien-6-one (ST-2: 2), 3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (ST-3: 3), and 3β,5α,6α-trihydroxyergosta-7,22
-diene (ST-4: 4) 。All were known compounds. From the isolates, ergosta-7,22-dien-3β-ol was isolated from P. sanguineus previously. The others were the first isolation from mycelial of P. sanguineus. Their structures were determined by 1H, 13C, 2D NMR, and MS spectral
analyses. The bioassay-guided fractionation of the crude ethanolic extract showed significant inhibition of LPS induced NO production in BV-2 cells up to a concentraction of 50 μg/ml for 87.5 %. EA-layer extract was also showed significant inhibition of LPS induced NO production in
BV-2 cells up to a concentraction of 20 μg/ml for 87.5 %. BuOH-layer extract of 20 μg/ml for inhibition of 45.5 %. Compound ST-1 of the inhibition at the concentraction of 15 μM in the value of 43.5 %.
謝誌………………………………………………………... 2
中文摘要…………………………………………………………… 4
英文摘要…………………………………………………………… 5
目錄………………………………………………………………… 7
圖表………………………………………………………………… 10
縮寫字全名對照…………………………………………………… 13
第一章 緒論………….……………………………………..…..… 14
1.1 研究背景………….………………………………………...….. 16
1.1.1 血紅密孔菌簡介………………..…………………….… 16
1.1.1.1 分類地位………………….….…………..…….…. 16
1.1.1.2 型態構造與分佈…………..……………..….…… 16
1.1.1.3 子實體外表型態…………………………………. 18
1.1.2 成分及生物活性………………..……..……..……….… 19
1.2 研究動機與目的…………………………………...……….…... 20
第二章 實驗結果………………………………..…………….…. 21
2.1 菌絲體及子實體之製備……..…………………………………. 21
2.1.1 菌絲體之培養………………………..………..………… 21
2.1.2 菌絲體外表型態….…………………..……………...….. 23
2.1.3 血紅密孔菌菌絲體生長曲線及多醣體、乙醇萃取物含
量之變化…………………………………………..…….
26
2.1.4 血紅密孔菌菌絲體與子實體乾重產率之比較….…….. 28
2.1.5 多醣體分析…………………………….………….….… 33
2.1.5.1 血紅密孔菌子實體多醣體之分子量………….... 33
2.1.5.2 血紅密孔菌菌絲體多醣體之分子量………….... 33
2.1.5.3 血紅密孔菌子實體多醣體之單醣組成……….... 34
2.1.5.4 血紅密孔菌菌絲體多醣體之單醣組成.………... 34
2.2 成份萃取、分離與結構解析………….……………….....…..... 41
2.2.1 萃取與分離……………………………………..……….. 41
2.2.2 結構解析………………………….……………..……… 44
2.2.2.1 化合物ST-1: ergosta-7,22-dien-3β-ol (1)……..………44
2.2.2.2 化合物ST-2: 3β,5α-dihydroxyergosta-7,22-dien-6-
one (2)……………………………….
61
8
2.2.2.3 化合物ST-3: 3β,5α,9α-tryhydroxyergosta-7,22-dien-
-6-one (3)……………………………
77
2.2.2.4 化合物ST-4: 3β,5α,6α-trihydroxyergosta-7,22-diene…
(4)…………………………………… 93
2.3 藥理作用…………………………………………………...…… 109
2.3.1 血紅密孔菌菌絲體之水層、乙酸乙脂層和正丁醇層萃
取物對LPS誘導BV-2 細胞抑制產生NO之作用…..….
109
2.3.2 血紅密孔菌菌絲體之乙酸乙脂層萃取物及其劃分Fr.
2-6 對LPS 誘導BV-2 細胞抑制產生NO 之作用………
111
2.3.3 血紅密孔菌菌絲體之正丁醇層萃取物及其劃分Fr. 1-5
對LPS 誘導BV-2 細胞抑制產生NO 之作用…………
113
2.3.4 化合物ST-1∼4 對LPS 誘導BV-2 細胞抑制產生NO 之
作用…………………………………………….………..
115
第三章 討論……………………………..……………….…….…. 117
第四章 實驗材料與方法…………………………..…………..…. 121
4.1 培養材料……………….…….………………................……… 121
4.1.1 菌株……………….…….…………..……….…..……… 121
4.1.2 儀器………………..…………….………………........… 121
4.2 培養方法………….………………………………………...….. 122
4.2.1 培養基之配製法……………………………..….……… 122
4.2.2 血紅密孔菌菌絲體培養法…..………………….……… 122
4.2.3 菌絲體生長指標………………………..………….…… 122
4.2.4 菌絲體萃取物之製備…………………....…………...… 123
4.2.5 抽提及分離…………….………………….……...…...... 123
4.2.6 多醣體分子量分佈分析…..…………………..…….….. 124
4.2.7 多醣體水解物之單醣組成分析…………..……….….... 125
4.2.8 統計方法…………………....…………………….....….. 126
4.3 菌絲體成份萃取、分離與結構解析…………………...….…… 127
4.3.1 溶媒及試藥……………..…………….....………….…… 127
4.3.2 管柱層析……..……………….………………..……...… 127
4.3.3 顯色劑…………………………………………………... 127
4.3.4 實驗儀器…………………..………….…………..….….. 127
4.4 實驗步驟………………………………………………….…..… 129
4.5 化合物之物理性質…………………………………………….. 131
ST-1: ergosta-7,22-dien-3β-ol (1)………..…………..…….….. 131
9
ST-2: 3β,5α-dihydroxyergosta-7,22-dien-6-one (2)…………... 131
ST-3: 3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (3)……. 132
ST-4: 3β,5α,6α-trihydroxyergosta-7,22-diene (4)………......… 133
4.6 藥理作用之評估方法…………………….……………..……… 134
4.6.1 BV-2 細胞培養………………..…………………….…… 134
4.6.2 MTT 分析試驗………….....…………….....….............… 134
4.6.3 NO 分析試驗………...…….…………....................……. 135
4.7 統計方法……………….……………………….…………….… 136
參考文獻……………………………………….……………...……. 137
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