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研究生:卜瑋彩
研究生(外文):Prokprae Puwavicheanchai
論文名稱:乾燥羅漢果萃取物之植化素及化學保護作用
論文名稱(外文):Phytochemical compounds and chemoprotective effect of dried Siraitia grosvenorii fruit extracts
指導教授:吳明昌邱亞伯
指導教授(外文):Ming-Chang WuAlbert Linton Charles
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:熱帶農業暨國際合作系
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:93
中文關鍵詞:羅漢果酚酸羅漢果皂苷抗氧化活性酒精傷害小鼠肝細胞
外文關鍵詞:Siraitia grosvenoriiLao Han Guophenolicsmogrosidesantioxidant activityalcoholic liver diseaseFL83B
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羅漢果,屬於葫蘆科的果實,生長於中國西南部,為中國傳統藥材之一,是可食用性的植物。經由美國食品藥物管理局通過,羅漢果萃取物被美國食品藥物管理局批准為一種公認安全的添加物。將乾燥羅漢果實的果肉及果皮以不同溶劑萃取(熱水及80%乙醇) 所得之萃取物;並將乾燥羅漢果實的果肉及果皮各用不同菌株(Saccharomyces bayanus 及 S. cerevisiae)進行發酵,分別探討不同部位的羅漢果萃取物、不同部位發酵的羅漢果和發酵時間對抗氧化能力、植化素成分及酒精誘導小鼠肝細胞(FL83B)損傷之保護能力。抗氧化能力包含DPPH自由基清除能力、鐵離子還原能力及還原力。利用RP-HPLC定性與定量萃取與發酵後樣品中的一種三萜苷類 (羅漢果皂苷 V)與六種多酚類(沒食子酸,咖啡酸,對香豆酸,兒茶素,芸香甘和槲皮素)。結果顯示,由熱水所萃取之乾燥羅漢果皮萃取物比起其他的萃取物含有較高的羅漢果皂苷 V、酚酸及類黃酮。然而經由S. bayanus發酵的羅漢果皮,其酚酸含量及抗氧化活性皆高於所有組別,且可降低酒精對細胞的傷害。細胞實驗發現,羅漢果萃取物在濃度1,000 μg/mL以下並不會造成小鼠肝細胞FL83B的毒性,而500 μg/mL 之羅漢果皮萃取物則具有最佳的酒精損傷小鼠肝細胞之保護能力,會有這些效果要歸功於羅漢果萃取物中含有羅漢果苷V、酚酸及黃酮類等抗氧化物質。因此,乾燥羅漢果萃取物可以當成一種天然保肝劑的成分,來抵抗酒精所產生的氧化壓力。
The fruits of Siraitia grosvenorii (Luo Han Kuo), belonging to family Cucurbitaceae, are traditional Chinese medicine and edible plant cultivated in south-western China. S. grosvenorii fruit extracts have been approved generally recognized as safe (GRAS) status by the Food and Drug Administration of the United States (USFDA). The effects of extraction solvents (hot water and 80% ethanol) in dried fruit flesh and peel extraction and effects of yeast strains (Saccharomyces bayanus and S. cerevisiae) and fermentation time in dried fruit flesh and peel fermentation were investigated for its antioxidant capacity and phytochemical components and hepatoprotective activity on ethanol injured mouse hepatocytes FL83B. The antioxidant activity was evaluated with 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability of plasma (FRAP), and reducing power assay. Quantitative and qualitative analysis of phytochemical compounds contained in extracts and fermented dried fruits were carried out by RP–HPLC revealed one triterpene glycoside (mogroside V) and six active phenolic compounds (gallic acid, caffeic acid, p-coumaric acid, catechin, rutin and quercetin). These results revealed that dried S. grosvenorii fruit peel in hot water extracts (PH) contained mogroside V, phenolic acids, flavonoids and exhibited higher than others extracts. However, fermented dried S. grosvenorii fruit peel by S. bayanus (PSB) showed the highest antioxidant activities and phenolics content compare to all samples, and also showed suppressed injured cell by alcohol. In Cytotoxicity assay, dried S. grosvenorii fruit extracts at the concentrations less than 1,000 μg/mL were considered to be non toxic to mouse hepatocytes FL83B and 500 μg/mL of dried S. grosvenorii fruit peel in hot water extract (PH) showed the most significant protective ability on ethanol injured cells. This ability may be related with antioxidant properties of dried S. grosvenorii fruit peel extract contributed by its compounds, which are mogroside V, phenolic acids and flavonoids. Hence, we proposed that dried S. grosvenorii fruit extract could potentially serve as hepatoprotective agent from natural source against alcohol-induced oxidative stress.
中文摘要 ............................................................................................................ I
English abstract ............................................................................................... III
Acknowledgements .......................................................................................... V
Table of Contents ............................................................................................ VI
List of Tables ................................................................................................... IX
List of Figures .................................................................................................. X
1. Introduction ................................................................................................... 1
2. Literature Review .......................................................................................... 3
2.1 Luo Han Guo ........................................................................................... 3
2.1.1 Botanical information ...................................................................... 3
2.1.2 Siraitia grosvenorii fruit .................................................................. 4 2.1.3 Pharmacological properties .............................................................. 5
2.2 Alcohol's effect on the liver .................................................................... 6
2.2.1 Free radicals ..................................................................................... 8
2.2.2 Ethanol-induced oxidative stress ................................................... 10 2.2.3 Reactive oxygen species-induced apoptosis .................................. 12 2.2.4 Antioxidant ..................................................................................... 12
2.2.5 Antioxidant against alcohol-induced liver disease ........................ 14
2.3 Color of the fruits .................................................................................. 15
2.4 Phenolic compounds ............................................................................. 16
2.4.1 Phenolic acids and aldehydes ......................................................... 17
2.4.2 Flavonoids ...................................................................................... 21 2.4.3 Tannin ............................................................................................ 24
2.5 Cucurbitane compounds ........................................................................ 25
2.6 Antioxidant assays ................................................................................ 29 2.6.1 DPPH (2,2-diphenyl-1-picrylhydrazyl) ......................................... 29
2.6.2 Ferric reducing ability of plasma ................................................... 29 2.6.3 Reducing power ............................................................................. 30
2.7 Cell viability .......................................................................................... 30
3. Materials and Methods ................................................................................ 32
3.1 Materials ................................................................................................ 32
3.1.1 Plant sample ................................................................................... 32
3.1.2 Cell line .......................................................................................... 33
3.1.3 Chemicals ....................................................................................... 33
3.2 Methods ................................................................................................. 34
3.2.1 Preparation of plant extracts .......................................................... 34
3.2.2 Preparation of fermented plant ....................................................... 34
3.2.3 L, a, b value .................................................................................... 35
3.2.4 pH value ......................................................................................... 35
3.2.5 Total soluble solids, TSS ............................................................... 35
3.2.6 Alcohol content .............................................................................. 35
3.2.7 Test of DPPH free radical scavenging activity .............................. 36
3.2.8 The ferric reducing ability of plasma (FRAP) ............................... 36
3.2.9 Reducing power assay .................................................................... 37
3.2.10 HPLC Analysis ........................................................................... 37
3.2.11 FL83B cell culture ....................................................................... 38
3.2.12 Cell viability assay ....................................................................... 39
3.2.13 Alcohol-injured cell test ............................................................... 40
3.2.14 Cell toxicity test ........................................................................... 40
3.2.15 Cell chemoprotective test ............................................................. 40
3.2.16 Cell morphology .......................................................................... 41
3.2.17 Statistical analysis ........................................................................ 41
4. Results and Discussion ................................................................................ 42
4.1 Physical characteristics analysis ........................................................... 42
4.1.1 L, a, b value .................................................................................... 42
4.1.2 pH value ......................................................................................... 45
4.1.1 Total soluble solids, TSS ............................................................... 46
4.1.2 Alcohol content .............................................................................. 47
4.2 Antioxidants capacity of dried S. grosvenorii fruits ............................. 49
4.2.1 Determination of DPPH Scavenging Activity ............................... 49
4.2.2 Determination of FRAP (ferric reducing ability of
plasma) assay ................................................................................. 52
4.2.3 Determination of reducing power .................................................. 54
4.3 HPLC Analysis ...................................................................................... 56
4.4 Alcohol-injured testing of FL83B cell line ........................................... 61
4.5 Cell toxicity testing of dried Siraitia grosvenorii fruit extracts ........... 64
4.6 Cell chemoprotective testing of dried Siraitia grosvenorii
fruit extracts .......................................................................................... 65
4.7 Analysis of correlations of mogroside V, phenolic acids,
flavonoids, antioxidants and FL83B chemoprotective effect ............... 68
5. Conclusions ................................................................................................. 71
6. References ................................................................................................... 72

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