第四章 研究結果
第三節 紅藜萃取物與其 rutin 對四氯化碳誘導肝損傷之影響
一、紅藜萃取物與對四氯化碳誘導肝損傷小鼠體重之影響
體重變化為生理評估的一種，當毒物進入生物體，會使生物體內代謝過程中 產生變化，進一步造成體重增加或減輕之情形 (Rajesh and Latha 2004)。本實驗 結果(表 4-7) 顯示，實驗初始 0 週時，各組體重均無差異 (p < 0.05)。實驗第 2
表 4-7 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠體重之影響
Table 4-7 The effects of red quinoa and its extracts on the body weight gain of the CCl4-induced liver injury mice
Groups
Body weight (g)
0th week 2nd week 4th week 6th week NOR 25.8 ± 1.4 a 26.4 ± 0.5 b 27.5 ± 1.8 b 29.0 ± 0.9 b
CCL 26.0 ± 1.5 a 24.5 ± 0.8 a 24.5 ± 3.4 a 26.5 ± 1.9 a HL-P 25.5 ± 1.6 a 25.0 ± 1.3 a 24.6 ± 2.2 a 26.5 ± 1.2 a HL-E 25.9 ± 1.5 a 24.4 ± 1.1 a 24.3 ± 2.8 a 26.6 ± 1.6 a HL-W 25.6 ± 1.6 a 24.8 ± 1.0 a 24.2 ± 1.5 a 26.0 ± 2.3 a Rutin 25.8 ± 1.5 a 24.4 ± 2.1 a 24.7 ± 3.3 a 26.0 ± 2.3 a
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4
induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
表 4-8 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠體重、肝重及肝重/體重比值 之影響
Table 4-8 The effects of red quinoa and its extracts on body weight, liver weight and liver weight/body weight ratio of the CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4
induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
Groups Liver weight (g) Body weight (g) Liver weight/Body weight (%)
NOR 1.38 ± 0.10 a 27.63 ± 1.41 d 5.00 ± 0.15 a CCL 2.00 ± 0.18 cd 25.50 ± 1.07 abc 7.87 ± 0.75 cd HL-P 1.82 ± 0.16 bc 24.63 ± 1.92 ab 7.39 ± 0.36 bc HL-E 2.20 ± 0.28 d 27.00 ± 2.20 d 8.11 ± 0.61 de HL-W 2.20 ± 0.35 d 25.75 ± 1.91 cd 8.51 ± 0.90 e
Rutin 1.65 ± 0.31 b 23.38 ± 3.25 a 7.04 ± 0.48 b
表 4-9 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠血清肝損傷指標 AST、ALT 與 ALP 含量之影響
Table 4-9 The effects of red quinoa and its extracts on serum AST, ALT and ALP activities of the CCl4-induced liver injury mice
NOR: normal group, CCL: CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice, fed red quinoa-powder, HL-E: CCl4 induced liver injury mice, fed red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice,fed red quinoa-water extracts, Rutin: CCl4 induced liver injury mice, fed rutin. Data are presented as mean
± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
Groups AST activity (U/L) ALT activity (U/L) ALP activity (IU/L) NOR 63.6 ± 4.2 a 41.3 ± 3.2 a 97.9 ± 10.0 c
CCL 230.0 ± 69.8 c 309.3 ± 31.6 d 84.3 ± 4.8 b HL-P 95.8 ± 9.3 a 215.0 ± 33.2 b 84.9 ± 4.9 b HL-E 174.6 ± 45.5 b 209.0 ± 36.9 b 76.8 ± 2.8 a HL-W 173.9 ± 60.9 b 226.8 ± 64.9 bc 82.5 ± 6.6 ab
Rutin 112.1 ± 19.6 a 264.1 ± 71.0 c 75.8 ± 6.9 a
低於 NOR 組，表示四氯化碳在代謝中會降低 ALP 活性。
表 4-10 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠血清肝臟合成指標總蛋白、白蛋白、球蛋白、白蛋白/球蛋白比值和總膽紅素之影 響
Table 4-10 The effects of red quinoa and its extracts on serum Total protein, Albumin, Globulin, A/G Ratio and total bilirubin of the CCl4-induced liver injury mice
NOR: normal group, CCL: CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice, fed red quinoa-powder, HL-E: CCl4 induced liver injury mice, fed red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice,fed red quinoa-water extracts, Rutin: CCl4 induced liver injury mice, fed rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
Groups Total protein (g/dL)
Albumin (g/dL)
Globulin
(g/dL) A/G Ratio TBIL
(mg/dL) NOR 5.46 ± 0.28 a 3.59 ± 0.16 a 1.85 ± 0.11 a 1.91 ± 0.06 a 0.04 ± 0.01 a CCL 7.40 ± 0.80 c 4.79 ± 0.22 c 2.51 ± 0.50 c 2.15 ± 0.17 b 0.12 ± 0.02 d HL-P 6.59 ± 0.16 b 4.38 ± 0.05 b 2.20 ± 0.15 bc 1.90 ± 0.11 a 0.08 ± 0.01 b HL-E 6.64 ± 0.29 b 4.43 ± 0.13 b 2.25 ± 0.18 bc 1.81 ± 0.08 a 0.08 ± 0.01 b HL-W 6.81 ± 0.69 b 4.46 ± 0.35 b 2.35 ± 0.38 bc 1.94 ± 0.26 a 0.10 ± 0.03 cd Rutin 6.30 ± 0.52 b 4.30 ± 0.25 b 2.04 ± 0.21 ab 1.83 ± 0.16 a 0.09 ± 0.01 ad
68
表 4-11 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠血清腎功能指標尿素氮和 肌酸酐之影響
Table 4-11 The effects of red quinoa and its extracts on serum BUN and CRE of the CCl4-induced liver injury mice
NOR: normal group, CCL: CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice, fed red quinoa-powder, HL-E: CCl4 induced liver injury mice, fed red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice,fed red quinoa-water extracts, Rutin: CCl4 induced liver injury mice, fed rutin. Data are presented as mean
± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
Groups BUN (mg/dL) CRE (mg/dL) NOR 23.33 ± 2.13 a 0.15 ± 0.02 a CCL 31.45 ± 5.39 b 0.25 ± 0.05 d HL-P 26.54 ± 5.77 ab 0.22 ± 0.06 cd HL-E 27.64 ± 4.02 ab 0.19 ± 0.03 ab HL-W 33.99 ± 12.69 b 0.21 ± 0.05 cd Rutin 32.85 ± 6.28 b 0.21 ± 0.04 cd
，表示四氯化碳誘導會造成腎臟損傷，但經由餵食詴驗物質與其活性成分 rutin，
圖 4-10 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠脂質過氧化之影響
Fig 4-10 The effects of red quinoa and its extracts on the liver lipid peroxidation of CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4
induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
圖 4-11 紅 藜 及 其 萃 取 物 對 四 氯 化 碳 誘 導 肝 損 傷 小 鼠 肝 臟 內 superoxide dismutase 活性之影響
Fig 4-11 The effects of red quinoa and its extracts on superoxide dismutase activity of CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
圖 4-12 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟內 Catalase 活性之影 響
Fig 4-12 The effects of red quinoa and its extracts on Catalase activity of CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
GPx 亦是抗氧化酵素之一，能藉由氧化 GSH 以分解 H2O2，進而避免氧化
圖 4-13 紅 藜 及 其 萃 取 物 對 四 氯 化 碳 誘 導 肝 損 傷 小 鼠 肝 臟 內 glutathione peroxidase 活性之影響
Fig 4-13 The effects of red quinoa and its extracts on glutathione peroxidase activity of CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4
induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
圖 4-14 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟內 glutathione 含量之 影響
Fig 4-14 The effects of red quinoa and its extracts on glutathione of CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c,d Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
圖 4-15 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟病理切片圖
Fig 4-15 The effects of red quinoa and its extracts on hepatic pathological changes of CCl4-induced liver injury mice. Magnification 100x. (black arrows undicates the position of liver injury).
(a) NOR: normal group (b) CCL : CCl4 induced liver injury mice (C) HL-P: CCl4
induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder (d) HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts (e) HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts (f) Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin
圖 4-16 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟病理切片圖
Fig 4-16 The effects of red quinoa and its extracts on hepatic pathological changes of CCl4-induced liver injury mice. Magnification 400x. (black arrows undicates the position of liver injury).
(a) NOR: normal group (b) CCL : CCl4 induced liver injury mice (C) HL-P: CCl4
induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder (d) HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts (e) HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts (f) Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin
圖 4-17 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟膠原染色圖 (Sirius red stain, 40X)
Fig 4-17 The effects of red quinoa and its extracts on hepatic collagen staining of CCl4-induced liver injury mice (Sirius red stain, 40X)
(a) NOR: normal group (b) CCL : CCl4 induced liver injury mice (C) HL-P: CCl4
induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder (d) HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts (e) HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts (f) Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin
圖 4-18 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟膠原染色圖 (Sirius red stain, 100X)
Fig 4-18 The effects of red quinoa and its extracts on hepatic collagen staining of CCl4-induced liver injury mice (Sirius red stain, 100X)
(a) NOR: normal group (b) CCL : CCl4 induced liver injury mice (C) HL-P: CCl4
induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder (d) HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts (e) HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts (f) Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin
十、紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟中細胞激素之影響 研究顯示
肝細胞損傷不是由誘導劑引起，而是受促發炎因子誘導發炎反應引起肝損傷 之結果 (Ramadori and Armbrust 2001)。四氯化碳代謝會活化巨噬細胞釋放細胞激 素 TNF-α 和 IL-6，使白血球凝集造成持續性的發炎反應，進一步造成肝纖維 化 (Bataller and Brenner 2005; Friedman 2004; Weber et al. 2003)。如圖 4-19 結果，
肝臟中促炎因子 TNF-α 和 IL-6 的含量，CCL 組顯著高於 NOR 組，表示四 氯化碳代謝提高發炎因子產生，而餵食紅藜萃取物紅藜粉末組、紅藜酒萃組、紅 藜水萃組和功效成分 Rutin 組與 CCL 組相比均顯著下降 (p<0.05)，顯示紅藜萃 取物能有效降低促發炎因子含量。
十一、紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟中纖維化因子之影響 TGF-β1 (transforming growth factor- β1) 是刺激肝星狀細胞分化成似肌纖維 母細胞，促進細胞外基質生成，抑制細胞外基質的分解，因此抑制或阻斷 TGF-β1 訊號/路徑可減少肝纖維化的程度 (Miyazaki et al. 2005)。結果示如圖 4-20，CCL 組比 NOR 組顯著提高 (p<0.05)，經由管餵紅藜萃取物紅藜粉末組、紅藜酒萃組、
紅藜水萃組和 Rutin 組與 CCL 組相比均顯著下降 (p<0.05)，表示紅藜萃取物能 抑制肝纖維因子 TGF-β1，進而達到改善。
α-SMA 是肝星狀細胞活化標誌物，因此抑制肝星狀細胞的活化是一個關鍵 點，以阻斷纖維化的進展。此結果顯示，如圖 4-21，長期肝損傷後，CCL 組顯 著高於 NOR 組 (p<0.05)，但管餵藜萃取物紅藜粉末組、紅藜酒萃組、紅藜水萃 組和 Rutin 組與 CCL 組相比均顯著下降 (p<0.05)，該結果證實紅藜萃取物均能 夠抑制的四氯化碳誘發活化星狀細胞，造成抑制膠原生長，降低膠原蛋白的累 積。
圖 4-19 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟內 TNF-α、IL-6 之影響 Fig 4-19 The effects of red quinoa and its extracts on TNF-α and IL-6 of CCl4-induced liver injury mice
TNF-α、IL-6 protein levels was measured with indirect ELTSA method. NOR:
normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin.
Data are presented as mean ± S.D. (n=6).a,b Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p <
0.05).
圖 4-20 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟內 TGF-β1 之影響 Fig 4-20 The effects of red quinoa and its extracts on TGF-β1 of CCl4-induced liver injury mice
TGF-β1 protein levels was measured with indirect ELTSA method. NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W:
CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=6).a,b, Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
圖 4-21 紅藜及其萃取物對四氯化碳誘導肝損傷小鼠肝臟內α-SMA 表現量之 影響
Fig 4-21 The effects of red quinoa and its extracts on α-SMA of CCl4-induced liver injury mice
NOR: normal group, CCL : CCl4 induced liver injury mice, HL-P: CCl4 induced liver injury mice fed 5.13 g/kg (rutin 8.46 mg/kg) of red quinoa-powder, HL-E: CCl4
induced liver injury mice fed 1.54 g/kg (rutin 16.4 mg/kg) of red quinoa- ethanol extracts, HL-W: CCl4 induced liver injury mice fed 1.54 g/kg (rutin 3.92 mg/kg) of red quinoa-water extracts, Rutin: CCl4 induced liver injury mice fed 16.4 mg/kg of rutin. Data are presented as mean ± S.D. (n=8).a,b,c, Different letters indicate significantly different values according to a one-way ANOVA with Duncan's multiple test (p < 0.05).
第五章 討論
膳食纖維可以促消化、增加飽食感，進而減少體重 (Jimenez-Cruz et al. 2006)，因此也是影響老鼠體重下降原因之一。
二、詴驗物質與其成分 rutin 對酒精性肝損傷小鼠肝臟氧化壓力之影響
酒精除了會透過 ADH 路徑代謝，同時也會利用 MEOS 氧化代謝系統和 CAT 路徑協助代謝，產生氧化壓力，而氧化壓力為誘發酒精性肝損傷之主要原 因之一 (Hoek and Pastorino 2002; Lu and Cederbaum 2008)。並代謝的過程中產生 大量的自由基，使與體內的抗氧化系統之間帄衡失調，干擾細胞氧化還原狀態，
酒精除了會透過 ADH 路徑代謝，同時也會利用 MEOS 氧化代謝系統和 CAT 路徑協助代謝，產生氧化壓力，而氧化壓力為誘發酒精性肝損傷之主要原 因之一 (Hoek and Pastorino 2002; Lu and Cederbaum 2008)。並代謝的過程中產生 大量的自由基，使與體內的抗氧化系統之間帄衡失調，干擾細胞氧化還原狀態，