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研究生:李炫璋
研究生(外文):Shiuann-Chang Lee
論文名稱:牛樟芝菌絲體之體內保肝功能評估及其熱水萃物在體外對基質金屬蛋白水解酶活性之影響
論文名稱(外文):Studies of the mycelium of Antrodia camphorata:In vivo protection against CCl4-induced liver damage and its aqueous extract’s effect in matrix metalloproteinases in vitro
指導教授:胡淼琳
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:88
中文關鍵詞:樟芝四氯化碳慢性肝損傷基質金屬蛋白熱水萃取物
外文關鍵詞:Antrodia camphorataCCl4chronic liver damagematrix metalloproteinaseSK-Hep-1 cellaqueous extract
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樟芝(Antrodia camphorata),僅生長在台灣特有的牛樟樹(Cinnamomum kanehirae)上,因其數量稀少且久為民間所流傳之良好民俗偏方,遂成為台灣市場上最昂貴的野生真菌。有鑑於肝病為我國之國病,久居國人十大死因之一,因此對於保肝防癌之健康食品開發實屬當務之急,故本論文之研究目的除了評估樟芝菌絲體在體內之保肝效果外,亦針對其熱水萃取物在體外模式中對SK-1肝癌細胞株所分泌之基質金屬蛋白水解酶活性之影響進行探討。期望樟芝菌絲體對於肝臟不僅具有保健之功效,對已罹患肝癌在化療後之癌細胞轉移抑制現象亦有所助益。
本研究之第一部分係研究樟芝菌絲體對CCl4所引起之肝傷害。其模式為以Wistar品系大鼠,分為A:control 組、B:CCl4處理組、C: CCl4+Silymarin組、D: CCl4+樟芝菌絲體低劑量組(0.25 g/kg)、E: CCl4+樟芝菌絲體中劑量組(0.5 g/kg)與F: CCl4+樟芝菌絲體高劑量組(1.25 g/kg)共為期八週。20% CCl4劑量為1.5 ml/kg,每週兩次。試驗結果顯示A組之GOT、GPT於八週間皆顯著低於B、C、D、E及F組,此外除第八週外,B、C、D、E、F五組間之GOT、GPT皆隨著時間的增加而上升,且各組間在第一及第三週時有顯著差異,顯示C組(silymarin組)與三組實驗組:D組(樟芝菌絲體低劑量組:0.25 g/kg b.w.)、E組(樟芝菌絲體中劑量組:0.5 g/kg b.w.)與F組(樟芝菌絲體高劑量組:1.25 g/kg b.w.)能有效的降低肝損傷時所造成的酵素脫逸,這個結果顯示樟芝菌絲體能顯著降低CCl4所誘導的急性肝損傷,且可降低多重CCl4所誘導的慢性肝傷害。此外包括抗氧化酵素(Catalase、SOD或GSH-Px)與TBARs值含量的結果顯示:silymarin與樟芝菌絲體處理組皆可增加抗氧化酵素含量,且降低脂質過氧化的程度。另外由肝、腎組織切片所進行的毒理觀察亦與生化值、各種傷害指標吻合,表示silymarin與中、高劑量樟芝菌絲體於此模式下能顯著的達到保肝效果。
樟芝傳統療效中有解毒、治癌、抗癌的功能。本研究第二部分即以樟芝菌絲體熱水萃取物在體外模式中對SK-1肝癌細胞株所分泌之基質金屬蛋白水解酶活性之影響。發現樟芝菌絲體熱水萃取物在10 mg/ml劑量下對於SK-Hep-1肝癌細胞株中所分泌的MMP-2及MMP-9,可顯著地抑制其活性,推測可能是和含有高量多醣體有關。然而其中可能的抑制機轉是值得我們再深入探討研究的。另外本研究亦以兩株癌化程度不同的細胞,比較樟芝菌絲體熱水萃取物對此兩株細胞是否具有不同程度的抗氧化效果。結果發現樟芝菌絲體熱水萃取物對癌化程度較低的Chang liver cell及癌化程度較高的SK-1 cell,在DNA傷害及脂質過氧化上並無差異,證實樟芝菌絲體熱水萃取物對於癌化程度不同之細胞皆具有相同的保護效果。在MTT assay的結果中亦發現1 mg/ml樟芝菌絲體熱水萃取物會造成SK-1細胞的死亡,但對Chang liver cell卻不會。因此進一步進行細胞週期分析,並證實1 mg/ml樟芝菌絲體熱水萃取物的確會造成SK-1細胞有大量的sub G1群落(細胞死亡)發生,但在chang liver cell卻無此現象。綜合以上研究分析,證實樟芝菌絲體在保健食品的應用上,的確具有開發潛力,且對於治療或預防癌症的復發及轉移上有相當助益。
Antrodia camphorata is a well-known fungus that only grows on Cinnamomum kanehirae and is only found in Taiwan with limited quantity. It is believed that A. camphorata has various beneficial effects including detoxification, anticancer, and protection against alcohol-induced liver diseases. Thus it has become the most valuable fungus in Taiwan. The liver disease is known as the national disease in Taiwan because about 15∼20% of the population in Taiwan have liver diseases, and every year approximately five thousand people die of liver cancer and four thousand of liver sclerosis. Therefore, it is important to develop health food, especially natural, unique materials, such as Antrodia camphorata in Taiwan for prevention and cure of liver disease.
The aim of this study was to investigate whether Antrodia camphorata is a good remedy for liver diseases. In this study, we used CCl4 to induce chronic liver damage in rats. Seventy-two Wistar rats were divided into six groups: Control, CCl4(negative control), CCl4+200 mg/kg silymarin(positive control), CCl4+0.25 g/kg mycelium of A. camphorata , CCl4+0.5 g/kg mycelium of A. camphorata, CCl4+1.25 g/kg mycelium of A. camphorata. CCl4 was given twice a week at a dose of 1.5 ml/kg of 20% CCl4 for 8 weeks. Damage to liver and kidney was determined by measuring serum levels of GOT, GPT at the first, third, sixth and eighth weeks. Levels of antioxidant enzymes (catalase, SOD, GSH-Px), lipid peroxidation and H.E. stain of liver were measured at the eight weeks, when rats were sacrifice. Chronic treatment with CCl4 induced hepatic damage, as indicated by elevated GOT and GPT levels and by the image of hepatic histopathology slices (H.E stain). The serum levels of GOT and GPT decreased markedly at week 8, suggesting severe liver damage at that time, as indicated by reduced protein levels. However, feeding of A. camphorata significantly decreased GOT and GPT activities and prevented structural damage to liver and kidney induced by CCl4, suggesting reduced severity of liver damage by feeding of A. camphorata. The effects of A. camphorata were stronger at the middle and high doses than at the low dose. Chronic treatment with CCl4 decreased antioxidant enzyme activities but increased TBARS in the liver. Feeding of A. camphorata significantly restored the activities of these enzymes and inhibited TBARS. The result indicates that feeding of A. camphorata can protect liver from free radical damage. Overall, our studies show that feeding rats with Antrodia camphorata significant protects against chronic liver damage induced by CCl4 intoxication. The effects are generally dose-dependent and neither Antrodia camphorata nor silymarin produces overt toxicities at the doses employed.
Recently, A. camphorata was further demonstrated to have an anticarcingenic effect in mice. In the second part, we studied the effect of the aqueous extract of A. camphorata mycelium on MMP-2 and MMP-9 released from SK-1 cells in vitro. A highly invasive SK-1 cell line of human hepatocellular carcinoma(HCC) was selected for this study. Gelatin-based zymography was adapted to assay the secretion of matrix metaloproteinase (MMP).We found that the A. camphorata aqueous extract inhibited the activity of metastasis protein MMP-2 and MMP-9. The results suggest that reduction of MMP-2 and MMP-9 may retard the invasive activity of carcinoma cell. In addition, we compare the effects of A. camphorata on DNA damage and lipid peroxidation exerted by Fe/NTA in both Chang liver cell and SK-1 cells. The results showed that there was no difference in the effect of A. camphorata on lipid peroxidation and DNA damage induced by Fe-NTA between Chang liver cells and SK-1 cells. We also found that treatment of SK-1 cells, but not Chang liver cells, with 1 mg/ml A. camphorata in SK-1 cell caused strong cytotoxicity. Further studies in cell cycle analysis by flow cytometry showed that A. camphorata aqueous extract at 1 mg/ml halted the cell cycle of SK-1 cells at the sub G1 phase but did not affect the cell cycle of Chang liver cells.
In summary, the results of this study illustrate that mycelium of A. camphorata has good liver-protective effects in vivo and exhibits strong antimetastatic effect in vitro. Further research related to the effects of mycelium of A. camphorata on liver damage and anti-cancer properties will be continued, so that the application of A. camphorata can be expanded.
總目錄
第一章:牛樟芝菌絲體之體內保肝功能評估……………………1
前言………………………………………………………………….3
材料與方法………………………………………………………....9
結果與討論…………………………………………………….…..16
結論………………………………………………………………...21
圖表…………………………………………………………………22
參考文獻…………………………………………………………....43
第二章:牛樟芝菌絲體熱水萃物在體外對SK-1肝癌細胞株所分泌之基質金屬蛋白水解酶活性之影響………………...……47
前言…………………………………………………………………50
材料與方法………………………………………………..……….56
結果與討論…………………………………………………………63
結論…………………………………………………………………68
圖表…………………………………………………………………69
參考文獻……………………………………………………………76
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