臺灣博碩士論文加值系統

摘要

由最近的研究發現，水產貝類具有抗腫瘤等生理活性，如文蛤、牡蠣及鮑魚等具有抗肝癌、胃癌、抗病毒及提高免疫力的作用；另外蜆被認為有保肝作用，民間常以熬煮蜆服食作為肝病治療，目前已有研究證實蜆肉水萃取物具有抑制人類肝癌細胞增生及誘導發生凋亡的效果。雖然研究已證實貝類具有抗腫瘤等生理活性，但其抗癌物質卻鮮少被發現，因此本研究目地為：1. 蜆萃取物抗癌的有效成分之分離及純化、2. 有效物質的化學結構鑑定。
初步研究發現台灣蜆以MeOH 萃取後再以EtOAc/H2O 進行分層萃取之FC-Me-EA，可造成人肝癌細胞(Hep-J2)凋亡。因此研究將甲醇萃取後EtOAc 分層的FC-Me-EA，以管柱層析(Al2O3, SiO2)、薄層層析以及高效液相層析等，進行分離純化，所得的各層，以人類肝癌細胞(6KK、HepG2)以及血癌細胞(HL60)培養，依所得抗癌效果bioactive-guide，進行分離純化，得抑制 HepH2 生長效果佳(4μg/ml)之112-5 fraction 由於所分離之化合物其產率太低，再進行大量製備後，由 NMR、MS 等進行化學結構評估，目前分離純化所得之 LWR-200-7為易分解之物質，初步鑑定為具 sterol 骨架的化合物。

Abstract

Recently, the marine shellfish have shown to inhibit cancer proliferation and other bioactivities. Hard calm, oyster and the abalone have been reported that could suppress the growth of liver carcinoma and gastric carcinoma, as well as antiviral and immunomodulated activities. In addition, local people believe that the soup made with freshwater clam have hepatoprotective activity. Although the freshwater calm aqueous extract has demonstrated to inhibit the proliferation of human liver carcinoma and induce the apoptosis of carcinoma and possess significant hepatoprotective activity, the study of bioactive compounds was not extensive. The goal of this study was to search the bioactive compound based on the anti-cancer activity-guided approach.
In the preliminary study, the ethyl acetate fraction after partition with water of freshwater calm methanolic extract induced apoptosis of Hep-J2 cell line. FC-Me-EA was further purified with column chromatography including Al2O3, SiO2 column chromatography and reverse phase high performance liquid chromatography based on bioactivity-guided approach. Different cell-lines including 6KK, HepG2 (human liver hepatoma) and HL-60 (human premyelocytic leukemia) were employed to study the cytotoxicities of fresh-clam extract and used to guide to isolate active fraction. This study demonstrated that the lipophilic fraction of fresh-clam possessed anti-cancer activities. One of fraction 112-5 was demonstrated to have significant anti-hepatoma activity (4 μg/ml). After further purification, a pure compound 200-7 consisting sterol skeleton was obtained.

目錄
中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
壹、緒論……………………………………………………………….1
研究目的…………………………………………………………..2
貳、 文獻整理………………………………………………………..3
ㄧ、貝類之藥用. …………………………………………………3
1. 文蛤藥用價值…………………………………………..4
2. 牡蠣藥用價值…………………………………………..5
3. 蜆的藥用價值…………………………………………..6
二、貝類具抗癌的有效物質…………………………………….7
1. 水產貝類之活性物質………………………………….7
2. Spisulosine的抗癌細胞生長能力………………………8
3. 作用機制……………………………………………….10
三、保肝作用：…………………………………………………12
1. 肝臟生理功能………………………………………….13
2. 四氯化碳引起肝損傷………………………………….14
3. 肝功能指數： ALT 及 AST…………………………14
4. 抗氧化測定 TBARs…………………………………15
5. Silymarin 肝炎之治療藥物…………………………15
四、Sterol 之細胞毒性………………………………………..16
参、實驗方法與材料……………………………………………….22
ㄧ、材料………………………………………………..……..22
（一） 實驗材料：………………………………………..22
（二） 化學藥品…………………………………………..22
（三） 儀器設備…………………………………………..23
二、實驗方法…………………………………………..…….25
（一）蜆之萃取及分離………………………………….25
（二）產率與純度之測定……………………………….26
（三）有效成分之純化分離及分析……………………26
（四）TLC 之呈色反應………………………………..30
（五）化合物之化學結構鑑定…………………………32
（六）活性評估：………………………………………33
1. 細胞培養…………………………………………34
2. 細胞存活率………………………………………34
3. 蜆萃取物之肝癌細胞活性評估…………………34
肆、結果與討論…………………………………………….35
（一）蜆之萃取…………………………………………35
（二）蜆萃取粗分離物抑制癌細胞生長之活性………35
（三）有效成分之分離、分析及其抗癌活性…………36
（四）蜆分離物之化學結構之評估……………………38
（五）結論
伍、參考文獻………………………………………………69
























圖目錄
表一、已知化合物 10、11、7 與研究分離所得 200-7 於13C-NMR 上位移訊號……………………………………………………………….42
圖一、實驗流程………………………………………………………..45
圖二、蜆萃取物分離後各層抑制人類血癌細胞 HL60 生長活性….46
圖三、蜆萃取物分離後各層抑制人類肝癌細胞 6KK 生長活性…..47
圖四、LWR-1-104-9 之 HPLC 圖譜…………………………….......48
圖五、93-3 管柱分離各層之 TLC 分析…………………………….49
圖六、 104-9 之分離…………………………………………………50
圖七、LWR-1-200-5 之 HPLC 圖譜………………………………..51
圖八、LWR-1-200-7 之 HPLC 圖譜………………………………..52
圖九、104-9 RP-HPLC 分離各層抑制人類肝癌細胞 HepG2 生長活 性………………………………………………………………53
圖十、200-7 分解後之 HPLC 分析………………………………..54
圖十一、LWR-1-212 管柱分離……………………………………..55
圖十二、LWR-1-212-3 分解後分析………………………………..56
圖十三、083-7分離各層抑制人類血癌細胞 HL60 生長活性…...57
圖十四、83-5~83-6 分離物抑制人類肝癌細胞 6KK 生長活性…60
圖十五、83-5~83-6 分離物抑制人類血癌細胞 HL60 生長活性..61
圖十六、LWR-1-96-3 之 HPLC 分析圖譜……………………….62
圖十七、096-3、-4分離物抑制肝癌細胞 HepG2 生長活性……63
圖十八、112-5 之 HPLC 分析圖譜…………………………….64
圖十九、112-5 之分離…………………………………………….65
圖二十、208-6之 HPLC 分析圖譜………………………………66
圖二十一、208-9之 HPLC 分析圖譜……………………………67
圖二十二、096-6之 HPLC 分析圖譜……………………………68

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