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研究生:張証維
研究生(外文):Cheng-Wei Chang
論文名稱:荔枝果實多酚萃取物具保護肝臟功能
論文名稱(外文):Hepatoprotective Activity on Ethanol-induced Injury by Polyphenolic Extracts From Litchi (Litchi chinensis Sonnerat)
指導教授:翁博群
指導教授(外文):Bor-Chun Weng
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物醫藥科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
中文關鍵詞:荔枝保肝保護修復原花青素酒精
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荔枝(Litchi chinensis Sonnerat)是中國特有的珍貴水果,於本草綱目記載利於保肝,但仍缺乏科學的證據;近期發現含有豐富的多酚類及黃酮類,並具有抗癌功能。我們於細胞毒性試驗證明台灣荔枝黑葉品種殼萃取成份對於小鼠多種細胞株,包括肝細胞BNL CL.2不具有毒性。利用高效液相層析儀(high performance liquid chromatography, HPLC)、核磁共振光譜儀(nuclear magnetic resonance, NMR)鑑定純化成分及流式細胞儀分析其對於肝細胞的生物活性,發現純化成分中原花青素類之proanthocyanidin A2與epicatechin可以強化恢復粒線體膜電位的能力,來保護肝細胞免受酒精毒性及自由基的傷害,此外經proanthocyanidin A2處理後,肝細胞株於酒精受損模式下,細胞週期中sub-G1的累積明顯減少,並提高了cyclin B, D以及CDK4蛋白質表現量,證明可以增加肝細胞數目,增加修復的速率。因此本篇研究利用酒精性肝損傷體外細胞模式,評估荔枝多酚萃取成分之保肝潛力,並進行確效成分純化分析,證明荔枝殼萃取成份中兩個主要純物質epicatechin和proanthocyanidin A2具有維持正常肝臟功能,保護酒精性的肝損傷,進而提出荔枝果實之殼具有良好潛力開發為治療酒精性肝臟疾病的食藥物,在臨床動物肝損傷實驗模式的探討也有迫切需要繼續完成以利相關產品的開發。
Alcoholism (chronic ethanol consumption) is one of primary medical problems with significant socio-economic consequence worldwide. Currently, silymarin from the milk thistle herb is the first-line drug used clinically on the alcohol liver disease (ALD). However, it has plenty side effects and uncertainties in curing some of liver diseases. Finding novel drug is in need to improve the efficiency on ALD therapy. Litchi is a precious tropical fruit originating from southern China enriched with phenolic compounds and flavonoids in its pericarp. It is traditionally documented as herbal medicine with hepatoprotective property. However, lack of scientific data to fulfill the wisdom of ancestral practice. In this study, we extracted and purified polyphenolic compounds of litchi pericarp. Cytotoxicity assay was performed for each fractionation of extract on various cell lines including the murine embryonic liver cell line (BNL CL.2). The model of ethanol induced cell injury in BNL CL.2 cells was adapted and showed litchi extracts were effectively protecting cells from ethanol induced reactive oxygen species (ROS). In addition, regenerative capacity of injured liver cells was analyzed. Using high performance liquid chromatography (HPLC) and NMR, two major compounds from litchi extracts, the epicatechin and proanthocyanidin A2, were identified, purification procedures were carried out subsequently. Flow cytometry analysis indicated the hepatoprotection of proanthocyanidin A2 was at least due to the strengthening mitochondria detoxification to ethanol induced excessive ROS. Moreover, there was a decrease percentage in the accumulated sub-G1 phase of ethanol treated liver cells and a faster regenerative proliferation with treatment of proanthocyanidin A2. To compare with silymarin, the litchi extracts had more protective effects on ethanol induced damages. In summary, we have discovered litchi pericarp extracts, possessing effective hepatoprotective properties on ethanol (EtOH)-induced liver cells BNL CL.2 injury. Two major compounds, epicatechin and proanthocyanidin A2 were identified in responsible for the results. Litchi pericarp may be promoted as complementary alternative medicines in ALD therapy.
目錄......................................................ii
表目錄....................................................vi
圖目錄...................................................vii
附圖目錄...................................................x
縮寫表...................................................xii
中文摘要..................................................xv
英文摘要.................................................xvi
一.文獻回顧................................................1
〈一〉肝臟之功能及其修復能力...............................1
1.肝臟角色和再生能力.......................................1
2.細胞週期與肝臟細胞再生能力之機制.........................3
〈二〉酒精性肝損傷與致病之機轉.............................5
1.酒精代謝和脂肪肝之形成...................................5
2.酒精抑制肝細胞之細胞週期.................................8
3.酒精造成肝細胞粒線體之功能喪失...........................9
〈三〉荔枝介紹和其生物活性................................11
〈四〉原花青素介紹和其生物活性............................14
二.文獻引用...............................................18
三.前言...................................................23
四.材料...................................................27
〈一〉材料來源............................................27
〈二〉細胞株..............................................27
〈三〉抗體................................................27
〈四〉化學試劑及藥品......................................28
〈五〉分離管柱及管柱填充劑................................30
〈六〉儀器設備............................................31
五.方法...................................................34
〈一〉黑葉荔枝殼成分萃取與分離............................34
1.萃取濃縮................................................34
2.管柱層析................................................34
〈二〉黑葉荔枝殼D40M劃分部成分之純化......................35
〈三〉細胞培養............................................36
1.BNL CL.2細胞培養........................................36
2.RAW 264.7細胞培養.......................................37
〈四〉荔枝殼劃分部毒性試驗................................37
1.MTT細胞毒性試驗.........................................37
2.LDH細胞毒性試驗.........................................38
〈五〉DCFH細胞內氧化壓力測試..............................39
〈六〉荔枝殼D40M劃分部保肝能力試驗........................40
1.傷肝前藥物處理..........................................40
2.MTT細胞存活試驗.........................................41
3.Trypan blue細胞存活試驗.................................41
〈七〉荔枝殼D40M劃分部修復能力試驗........................41
1.傷肝後藥物處理..........................................41
2.Alamar blue細胞修復試驗.................................42
3.Scratch wound healing assay物理性傷害修復試驗...........42
〈八〉荔枝成分epicatechin及proanthocyanidin A2修復酒精性傷害肝細胞試驗................................................43
1.粒線體膜電位檢測........................................43
2.粒線體螢光顯微鏡觀察....................................44
3.細胞週期分析試驗........................................44
4.細胞週期蛋白質分析......................................45
〈九〉統計分析............................................46
六.結果...................................................47
〈一〉荔枝殼粗萃取物及劃分部分離..........................47
〈二〉荔枝殼劃分部細胞毒性試驗............................47
〈三〉荔枝殼劃分部細胞內ROS含量檢測.......................48
〈四〉酒精引起肝損傷試驗測試荔枝殼D40M劃分部之保肝能力....50
〈五〉荔枝殼D40M劃分部對於肝細胞修復能力之影響............51
〈六〉荔枝殼D40M劃分部中有效成分epicatechin及proanthocyanidin A2之純化.................................52
〈七〉Epicatechin及proanthocyanidin A2修復能力試驗........53
1.酒精性傷害修復..........................................53
2.物理性傷害修復..........................................54
〈八〉Proanthocyanidin A2可改變粒線體膜電位...............54
〈九〉Proanthocyanidin A2加速酒精受損後細胞週期進行.......56
七.討論...................................................59
八.參考文獻...............................................69
結果圖....................................................73
附圖......................................................96
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