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研究生:張峻維
研究生(外文):Chun-Wei Chang
論文名稱:第一部分. 中國穿鞘花全草化學成分與抗結核活性之研究第二部分. 披覆炭團菌二次代謝產物與抗發炎活性之研究
論文名稱(外文):Part I. Chemical Constituents and Antitubercular Activity from the Whole Plant of Amischotolype hispidaPart II. Secondary Metabolites and Antiinflammatory Activity of an Endophytic Fungus, Hypoxylon investiens
指導教授:陳益昇陳益昇引用關係鄭銘仁鄭銘仁引用關係
指導教授(外文):Ih-Sheng ChenMing-Jen Cheng
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:天然藥物研究所
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:2013
畢業學年度:101
語文別:中文
論文頁數:271
中文關鍵詞:中國穿鞘花鴨跖草科抗結核病披覆炭團菌炭角菌科內生菌竹頭角木薑子抗發炎
外文關鍵詞:Amischotolype hispidaCommelinaceaeanti-tubercular activityHypoxylon investiensXylariaceaeendophytic fungusLitsea akoensis var. chitouchiaoensisLauraceaeanti-inflammatory
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Part I: 中國穿鞘花 (Amischotolype hispida (Less.& A. Rich.) Hong)為鴨跖草科 (Commelinaceae)植物,為多年生草本,分布於全島中低海拔之森林中。穿鞘花屬世界有20種,而台灣僅一種,而本植物全草之甲醇萃取物具有抗結核活性,且過去對於穿鞘花屬未曾有天然物分離及其相關活性之研究。
由乙酸乙酯可溶部具抗結核活性層利用活性導向劃分法得到共25個化合物,包含四個新化合物: amisbenzoic acid (1)、amisnolide (2)、amisphytin (3)與amislignol (4)及一個由天然界首次分離之lignan類化合物: (–)-glaberide I (5),以及20個已知化合物: veratic acid (6)、4-methoxybenzoic acid (7)、(–)-syringaresinol (8)、(±)-pinoresinol (9)、(+)-4-ketopinoresinol (10)、methyl palmitate (11)、methyl linoleate (12)、palmitic acid (13)、α-tocopherol (14)、γ-tocopherol (15)、δ-tocopherol (16)、ubiquinone Q9 (17)、α-tocopheryl quinone (18)、trans-phytol (19)、indole-3-carbaldehyde (20)、24-methylenecycloartanol (21)、cycloartenol (22)、β-sitosterol (23)、β-sitostenone (24)與ergosterol peroxide (25)。上述化合物皆由各種圖譜分析決定結構。
抗結核活性實驗中,palmitic acid (13)顯示有抗結核活性,其MIC為20.0 μg/mL,並也針對了四種不同市售長鏈類似物進行抗結核活性探討,其中比palmitic acid少了兩個碳的myristic acid,顯示較弱的抗結核活性,其MIC為50.0 μg/mL,因此推測其鏈長為14與16個碳,對於抗結核之活性是有其意義存在。
Part II: 披覆炭團菌 (Hypoxylon investiens)為台灣固有種植物之內生真菌,是從樟科竹頭角木薑子之莖部所分離出來,並以液態發酵培養之正丁醇粹取物以管柱層析法進行分離得到共29個化合物,包含三個新的化合物: hypoxyloamide (26)、8-methoxynaphthalene-1,7-diol (27)及hypoxylonol (28),七個化合物由天然物首次分離: investiamide (29)、hypoxypropanamide (30)、hypoxylonol A (31)、investienol (32)、2-heptylfuran (33)、(S)-5-methyl-8-O-methylmellein (34)及(R)-O-methylsclerone (35),以及19個已知化合物: indole-3-carbaldehyde (20)、ergosterol peroxide (25)、N-acetyltyramine (36)、nicotinamide (37)、8-methoxynaphthalen-1-ol (38)、1,8-dimethoxynaphthalene (39)、xylaranol B (40)、epi-guaidiol A (41)、(R)-5-hydroxymethylmellein (42)、(R)-8-O-methylmellein (43)、(R)-5-methylmellein (44)、(–)-regiolone (45)、xylarenone (46)、2-hydroxyphenethyl alcohol (47)、cyclo(L-Pro-L-Tyr) (48)、monascuspyrrole (49)、3-hydroxy-2-methyl-4H-pyran-4-one (50)、4-hydroxy-6-methyl-2H-pyran-2-one (51)與ergosta-4,6,8(14),22-tetraen-3-one (52)。上述化合物之結構皆由各種圖譜分析決定。
將足夠量之化合物進行抗發炎功效之篩選,測試在RAW 264.7細胞株中對於抑制脂多醣誘導之NO、IL-6及TNF-α所產生之能力。其中,8-methoxynaphthalene1,7-diol (27)、8-methoxynaphthalen-1-ol (38) 及1,8-dimethoxynaphthalene (39)具有強的抑制NO產生之活性,其IC50分別為11.8, 17.8及13.3 μM,且優於對照組quercetin (IC50 36.8 μM)。化合物27、38及39也顯示了強的抑制IL-6產生之活性,其IC50分別為9.2, 18.0及2.0 μM,且優於對照組quercetin (IC50 31.3 μM)。

Part I: Amischotolype hispida (Less.& A. Rich.) Hong (Commelinaceae) is a perennial herb, distributed over forest floors at low to middle altitudes throughout Taiwan. There are 20 species of Amischotolype genus around the world and only one species in Taiwan. The methanolic extract of the whole plant of this plant was shown with antitubercular activity. However, the phytochemistry and biological activities of Amischotolype genus have never been conducted.
Bioassay-guided fractionation of the active ethyl acetate-soluble layer has led to the isolation of four new compounds: amisbenzoic acid (1), amisnolide (2), amisphytin (3), amislignol (4) and one compound first isolated from nature, (–)-glaberide I (5), along with 20 known compounds, veratic acid (6), 4-methoxybenzoic acid (7), (–)-syringaresinol (8), (±)-pinoresinol (9), (+)-4-ketopinoresinol (10), methyl palmitate (11), methyl linoleate (12), palmitic acid (13), α-tocopherol (14), γ-tocopherol (15), δ-tocopherol (16), ubiquinone Q9 (17), α-tocopheryl quinone (18), trans-phytol (19), indole-3-carbaldehyde (20), 24-methylenecycloartanol (21), cycloartenol (22), β-sitosterol (23), β-sitostenone (24), and ergosterol peroxide (25). The structures of isolates were determined by spectroscopic techniques.
Palmitic acid (13) showed antitubercular activity with MIC value of 20.0 μg/mL and other four different lengths of fatty acid analogues such as myristic acid with less two methylenes than palmitic acid which showed weak antitubercular activity with MIC value of 50.0 μg/mL. From the biological results of the study, it is meaningful for antitubercular activity on the carbon numbers from 14 and 16 of saturated fatty acid analogues.
Part II: Hypoxylon investiens was an endophytic fungus, isolated from the stem of Taiwan endemic plant, Litsea akoensis Hayata var. chitouchiaoensis Liao (Lauraceae). H. investiens was processed through liquid fermentation. The n-BuOH extract of H. investiens was further chromatographed and led to the isolation of three new compounds, hypoxyloamide (26), 8-methoxynaphthalene-1,7-diol (27), and hypoxylonol (28), and ten compounds first isolated from nature, investiamide (29), hypoxypropanamide (30), hypoxylonol A (31), investienol (32), 2-heptylfuran (33), (S)-5-methyl-8-O-methylmellein (34), and (R)-O-methylsclerone (35), along with 19 known compounds, indole-3-carbaldehyde (20), ergosterol peroxide (25), N-acetyltyramine (36), nicotinamide (37), 8-methoxynaphthalen-1-ol (38), 1,8-dimethoxynaphthalene (39), xylaranol B (40), epi-guaidiol A (41), (R)-5-hydroxymethylmellein (42), (R)-8-O-methylmellein (43), (R)-5-methylmellein (44), (–)-regiolone (45), xylarenone (46), 2-hydroxyphenethyl alcohol (47), cyclo(L-Pro-L-Tyr) (48), monascuspyrrole (49), 3-hydroxy-2-methyl-4H-pyran-4-one (50), 4-hydroxy-6-methyl-2H-pyran-2-one (51), and ergosta-4,6,8(14),22-tetraen-3-one (52). The structures of isolates were determined by spectroscopic techniques.
The isolates with enough amounts were screened for their ability to inhibit NO, IL-6, and TNF-α production in LPS-activated RAW 264.7 cells. Among these isolates, 8-methoxynaphthalene-1,7-diol (27), 8-methoxynaphthalen-1-ol (38) and 1,8-dimethoxynaphthalene (39) showed the stronger NO inhibitory activity with IC50 values of 11.8, 17.8, and 13.3 μM than the positive control quercetin (IC50= 36.8 μM). Compounds 27, 38, and 39 also showed stronger IL-6 inhibitory activity with IC50 values of 9.2, 18.0, and 2.0 μM than the positive control quercetin (IC50= 31.3 μM).


目錄
目錄……………………………………………………………………………............…2
圖目錄……………………………………………………………………………..…5
表目錄…………………………………………………………………….……..…14
中文摘要…..………………………………………………………………………..…16
英文摘要…………………………………………………………..……………..…...17
Glossary of Abbreviations………………………………………………………..…..…18

第一章、序言
第一節、中國穿鞘花……………………………………………………….19
第二節、竹頭角木薑子及其內生真菌…………………………………….21
第二章、研究動機與目的
第一節、中國穿鞘花……………………………………………………….22
第二節、內生真菌二次代謝產物…………………………………..…..….24
第三章、過去文獻回顧
第一節、鴨跖草科植物成分之過去文獻回顧…………………..….……26
第二節、炭團菌屬真菌二次代謝產物過去文獻回顧…………....…….34
第四章、萃取與分離
第一節、中國穿鞘花……………………….………………………………48
第二節、披覆炭團菌……………………………….……………………...53
第五章、化合物之結構鑑定
第一節、amisbenzoic acid (1)之結構鑑定…………..………………......…..57
第二節、amishisnolide (2)之結構鑑定…………….……………......……..65
第三節、amisphytin (3)之結構鑑定……………………………...…....…..73
第四節、amislignol (4)之結構鑑定…………………………………...……..82
第五節、(–)-glaberide I (5)之結構鑑定…………….……………...…....…92
第六節、hypoxyloamide (26)之結構鑑定………………………………101
第七節、8-methoxynaphthalene-1,7-diol (27)之結構鑑定…….………...109
第八節、hypoxylonol (28)之結構鑑定…………….…………………….117
第九節、investiamide (29)之結構鑑定…………….………….………..127
第十節、hypoxypropanamide (30)之結構鑑定…………………….…..135
第十一節、hypoxylonol A (31)之結構鑑定………….……….………...143
第十二節、investienol (32)之結構鑑定…………….……….………….152
第十三節、2-heptylfuran (33)之結構鑑定……………….….………….161
第十四節、(S)-5-methyl-8-O-methylmellein (34)之結構鑑定…………169
第十五節、(R)-O-methylsclerone (35)之結構鑑定….…………………..177
第十六節、8-methoxynaphthalene-1-ol (38)之結構鑑定………………185
第十七節、1,8-dimethoxynaphthalene (39)之結構鑑定….…....……….191
第十八節、(R)-5-hydroxymethylmellein (42)之結構鑑定…………..…197
第十九節、cyclo(L-Pro-L-Tyr) (48)之結構鑑定………………...….…...203
第二十節、monascuspyrrole (49)之結構鑑定………………..………...210
第二十一節、3-hydroxy-2-methyl-4H-pyrone (50)之結構鑑定…..…...216
第二十二節、其他已知化合物之結構研究…………………………...219
第六章、生物活性
第一節、抗結核病活性之研究………………………………………......220
第二節、抗發炎活性之研究……………………………………………..222
第七章、結論………………………………………………….………………...…….223
第八章、實驗部分
第一節、儀器與材料…………………………………….………………..225
第二節、活性試驗法
第一部分、抗結核活性測試……………………………….228
第二部分、抗發炎活性測試…………………………….....230
第三節、物種來源、萃取與分離
第一部分、中國穿鞘花……………………..……………...….235
第二部分、H. investiens……….…..……………….................238
第五節、化合物之實驗數據……………………………….……………..244
第九章、參考文獻………………………………………………….………………….264


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