第四章 研究結果
第二節 探討 DOW 發酵 Cordyceps militaris 小鼠肝臟抗氧化、抗發炎及預
一、不同培養基質與水分對蛹蟲草發酵產物內腺苷及蟲草素含量之影響
研究指出不同培養基質會造成蛹蟲草發酵產物內腺苷與蟲草素含量不同 (Hung et al., 2015)，本研究首先以燕麥 (oat)、紅藜 (red quinoa)、白米 (rice) 及 薏仁 (barley) 作為發酵基質，培養 30 天後分析發酵產物內腺苷與蟲草素含量，
篩選適合之基質作為後續動物實驗試驗物質培養之基質，結果顯示以紅藜作為基 質之蛹蟲草發酵產物內腺苷與蟲草素含量，均顯著高於 (p<0.05) 其他基質發酵 之蛹蟲草發酵產物，而蟲草素含量則以薏仁作為基質之蛹蟲草發酵產物，高於其 他兩種基質之發酵產物。
DOW 已證實可有效提高微生物生長與代謝產物增加，因此以紅藜為培養基 質 DOW 作為水分來源，探討 DOW 能否有效增加蛹蟲草發酵產物內腺苷與蟲 草素含量，圖 4-8 與 4-9 結果得知 DOW 顯著提高蛹蟲草發酵產物內腺苷 (p<0.05) 與蟲草素 (p<0.01) 含量，對於紅藜作為基質之蛹蟲草發酵產物也具有 提高代謝產物之效果。
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圖 4-6 不同基質對蛹蟲草中腺苷含量之影響
Figure 4-6 Effect of different substrates on the adenosine content of C. militaris .Data are presented as mean ± SD (n=3). ** indicated the significant difference (p<0.001) as compared with Red Quinoa.
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圖 4-7 不同基質對蛹蟲草中蟲草素含量之影響
Figure 4-7 Effect of different substrates on the cordycepin content of C. militaris.
Data are presented as mean ± SD (n=3). *,**, *** indicated the significant difference (p
< 0.05, p < 0.01 and p < 0.001, respectively) as compared with red quinoa.
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圖 4-8 DOW 對蛹蟲草發酵紅藜之發酵物內腺苷含量之影響
Figure 4-8 Effect of different substrates on the adenosine content of DCM–RQ.
UCM–RQ: UPW–CM cultured with red quinoa, DCM-RQ: DOW–CM cultured with red quinoa. Data are presented as mean ± SD (n=3). ** indicated the significant difference (p<0.01) as compared with UCM–RQ.
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圖 4-9 DOW 對蛹蟲草發酵紅藜之發酵物內蟲草素含量之影響
Figure 4-9 Effect of DOW on the cordycepin content of DCM-RQ. UCM-RQ: UPW–
CM cultured with red quinoa, DCM–RQ: DOW–CM cultured with red quinoa.Data are presented as mean ± SD (n=3). * indicated the significant difference (p<0.05) as compared with UCM–RQ.
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二、小鼠體重變化
過去研究顯示酒精誘發動物肝損傷模式中，餵食酒精之組別體重增加量會顯 著下降 (Duly et al., 2015)，本研究中餵食酒精液態飼料之 EtOH 誘發組體重雖 有緩慢增加，但於第四週時體重已明顯低於 Nor 組，且隨著餵食時間增加體重 逐漸下降。餵食 DCM-RQ 組體重隨時間逐漸增加，且於第四週 DCM-RQ 組體 重已顯著高於 EtOH 誘發組 (p<0.001)，而 UPW-RQ、CC、AS 及 DOW 之組 別體 重均 隨時間逐 漸增加 ， 與 DCM-RQ 組體重 並無顯著 差異 (p>0.05) ， DOW-CM 可改善酒精飲食導致的體重下降。
三、DOW 發酵蛹蟲草對酒精性肝損傷小鼠體重、肝重與肝重/體重比值之影響 過去研究結果顯示酒精容易造成體重下降、肝重增加以及肝重/體重比值上 升 (Clugston et al., 2011)，本實驗結果可得知餵食酒精液態飼料之 EtOH 組體重 顯著低於 Nor 組 (p<0.001)，雖然 EtOH 組肝重顯著低於 Nor 組 (p<0.001)，
但肝重/體重之比值則較高於 Nor 組，而 DCM-RQ 組雖然在體重顯著高於 EtOH 組 (p<0.05)，但肝重/體重之比值則顯著低於 EtOH 組 (p<0.01)，其他試 驗物質組在體重方面與 DCM-RQ 組並無顯著差異 (p>0.05)，可是肝重略高於 DCM-RQ 組，因此肝重/體重比值雖有下降效果但不如 DCM-RQ 組顯著。
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表 4-5 DOW 培養之蛹蟲草發酵產物對小鼠體重之影響
Table 4-5 The effect of C. militaris-fermented products cultured with DOW on body weight of alcohol induce liver injury mice
groups
body weight (g)
0th week 2nd week 4th week 6th week
Nor 28.38 ± 1.41＃＃＃ 27.38 ± 1.11＃＃＃ 29.50 ± 0.50＃＃＃, ＊＊＊ 31.38 ± 0.70＃＃＃, ＊＊＊
EtOH 24.88 ± 1.27 25.50 ± 0.50 24.13 ± 1.05 25.75 ± 0.66 DCM-RQ 25.88 ± 1.17 25.50 ± 1.22 26.63 ± 0.86＃＃＃ 28.13 ± 0.93＃＃＃
UCM-RQ 25.38 ± 1.32 24.63 ± 0.48 26.25 ± 0.66＃＃＃ 28.00 ± 0.87＃＃＃
CC 26.00 ± 0.87 25.38 ± 1.41 26.50 ± 1.22＃＃＃ 27.13 ± 1.27 AS 25.88 ± 0.93 24.88 ± 0.93 26.38 ± 0.86＃＃＃ 28.38 ± 1.22＃＃＃
DOW 26.13 ± 1.54 26.50 ± 1.22 26.63 ± 0.48＃＃＃ 27.13 ± 0.60 Nor: normal group, EtOH: alcohol-induced liver injury mice, DCM-RQ: alcohol induced liver injury mice, fed DOW-CM-red quinoa, UCM-RQ: alcohol induced liver injury mice, fed UPW-CM-red quinoa, CC: alcohol induced liver injury mice, fed cordycepin, AS: lcohol induced liver injury mices,fed adenosine, DOW: alcohol induced liver injury mice, fed DOW.
Data are presented as mean ± SD (n=8). ＃,＃＃,＃＃＃ indicated the significant difference (p <
0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH. *,**, ***
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ.
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表 4-6 DOW 培養之蛹蟲草發酵產物對小鼠體重、肝重及肝重/體重比值之影響 Table 4-6 The effect of C. militaris-fermented products cultured with DOW on body weight, liver weight and liver weight/body weight ratio of alcohol induce liver injury mice
groups Body weight Liver weight Liver weight/Body weight ratio (%) Nor 26.75 ± 1.30＃＃＃, ＊＊＊ 0.99 ± 0.03＃＃＃, ＊＊＊ 3.70 ± 0.11 EtOH 23.00 ± 1.22＊ 0.88 ± 0.04 3.83 ± 0.14＊＊
DCM-RQ 24.75 ± 1.20＃ 0.88 ± 0.04 3.57 ± 0.13＃＃
UCM-RQ 23.50 ± 1.12 0.89 ± 0.06 3.77 ± 0.17＊ CC 23.63 ± 1.58 0.89 ± 0.06 3.77 ± 0.15＊ AS 24.38 ± 1.41 0.90 ± 0.04 3.68 ± 0.16 DOW 24.50 ± 1.58 0.90 ± 0.06 3.68 ± 0.13 Nor: normal group, EtOH: alcohol-induced liver injury mice, DCM-RQ: alcohol induced liver injury mice, fed DOW-CM-red quinoa, UCM-RQ: alcohol induced liver injury mice, fed UPW-CM-red quinoa, CC: alcohol induced liver injury mice, fed cordycepin, AS: alcohol induced liver injury mice, fed adenosine, DOW: alcohol induced liver injury mice, fed DOW. Data are presented as mean ± SD (n=8). ＃,＃＃,＃＃
＃ indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ.
76 AST 活性 (p<0.001)，DCM-RQ (p<0.001)、UCM-RQ (p<0.01)、CC (p<0.01)、AS (p<0.05) 及 DOW (p<0.05) 組能顯著降低 ALT 含量，DCM-RQ (p<0.001)、
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六、DOW 發酵蛹蟲草對酒精性肝損傷小鼠肝臟內脂質含量之影響
酒精可導致大量脂質生成並堆積於肝臟內 (Louvet and Mathurin, 2015)，餵 食酒精八周後可觀察到 EtOH 組肝臟內 TC 及 TG 均顯著高於 (p<0.001) Nor 組， DCM-RQ、UCM-RQ、CC、AS 及 DOW 組之小鼠肝臟內 TC 均顯著高 於 EtOH 組 (p<0.001)，其中 UCM-RQ (p<0.01) 與 DOW (p<0.001) 組小鼠肝 臟內 TC 更顯著高於 DCM-RQ 組，而小鼠肝臟內 TG 分析結果顯示 DCM-RQ 及 UCM-RQ 組肝臟內 TG 含量與 EtOH 組並無顯著差異 (p>0.05)，至於 CC (p<0.05)、AS (p<0.001) 及 DOW (p<0.01) 組肝臟內 TG 含量均顯著高於 EtOH 組，因此試驗物質均無法有效降低肝臟內脂質含量。
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表 4-7 DOW 培養之蛹蟲草發酵產物對小鼠血清生化值 AST、ALT、ALP、TC 與 TG 之影響
Table 4-7 The effect of C. militaris-fermented products cultured with DOW on serum AST、ALT、ALP、TC and TG of alcohol induce liver injury mice
AST (U/L) ALT (U/L) ALP (U/L) TC (mg/dL) TG (mg/dL) Nor 48.38 ± 2.23＃＃＃,＊＊＊ 16.13 ± 1.45＃＃＃ 74.38 ± 9.16＃＃＃ 107.13 ± 7.88＃＃＃, ＊ 89.13 ± 12.91＃＃＃
EtOH 58.63 ± 2.87＊＊＊ 23.50 ± 2.06＊＊＊ 91.63 ± 5.68＊＊＊ 125.50 ± 8.08＊ 154.50 ± 12.28＊＊＊
DCM-RQ 41.25 ± 1.64＃＃＃ 16.00 ± 1.41＃＃＃ 78.13 ± 4.88＃＃＃ 116.88 ± 6.73＃ 82.50 ± 6.56＃＃＃
UCM-RQ 47.13 ± 1.96＃＃＃,＊＊＊ 19.63 ± 2.29＃＃,＊＊ 73.38 ± 3.20＃＃＃ 121.63 ± 6.71 107.00 ± 5.48＃＃＃,＊＊＊
CC 49.88 ± 2.42＃＃＃,＊＊＊ 19.88 ± 2.09＃＃,＊＊ 74.75 ± 8.00＃＃＃ 124.38 ± 6.54＊ 109.13 ± 5.58＃＃＃,＊＊＊
AS 51.38 ± 4.30＃＃＃,＊＊＊ 20.88 ± 3.06＃＃,＊＊＊ 81.00 ± 5.57＃＃ 128.50 ± 7.09＊＊ 111.38 ± 6.28＃＃＃,＊＊＊
DOW 50.13 ± 3.10＃＃＃,＊＊＊ 21.00 ± 1.80＃＃,＊＊＊ 84.63 ± 7.26 114.88 ± 4.94＃ 95.88 ± 5.37＃＃＃,＊＊＊
Nor: normal group, ETOH: alcohol-induced liver injury mice, DCM-RQ: alcohol induced liver injury mice, fed DOW-CM-red quinoa, UCM-RQ: alcohol induced liver injury mice, fed UPW-CM-red quinoa, CC: alcohol induced liver injury mice, fed cordycepin, AS: alcohol induced liver injury mice, fed adenosine, DOW: alcohol induced liver injury mice, fed DOW. Data are presented as mean ± SD (n=8). ＃,＃＃,＃＃＃ indicated the significant difference (p <
0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ.
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表 4-8 DOW 培養之蛹蟲草發酵產物對小鼠肝臟內 TC 與 TG 之影響
Table 4-8 The effect of C. militaris-fermented products cultured with DOW on liver TC and TG of alcohol induce liver injury mice
TC (mg/g) TG (mg/g)
Nor: normal group, EtOH: alcohol-induced liver injury mice, DCM-RQ: alcohol induced liver injury mices,fed DOW-CM-red quinoa, UCM-RQ: alcohol induced liver injury mice, fed UPW-CM-red quinoa, CC: alcohol induced liver injury mice, fed cordycepin, AS: alcohol induced liver injury mice, fed adenosine, DOW: alcohol induced liver injury mice, fed DOW.
Data are presented as mean ± SD (n=8). ＃,＃＃,＃＃＃ indicated the significant difference (p <
0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH *,**, ***
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ.
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圖 4-10 DOW 培養之蛹蟲草對酒精性肝損傷小鼠肝臟病理變化之影響
Fig. 4-10 The effects of C. militaris-fermented products on hepatic pathological changes of the alcohol induced liver injury mice.
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果劣於 DCM-RQ 組。
GPx 為生物體內之抗氧化酵素，可藉由氧化 GSH 以分解 H2O2 避免氧化 壓力累積 (Zachariah et al., 2014)，本實驗結果顯示 EtOH 組小鼠 (p<0.05) 長期 飲用酒精使得肝臟內 GPx 活性與 Nor 組相比活性顯著降低，可是 UCM-RQ 組與 EtOH 組 (p<0.001) 相比則可顯著預防 GPx 活性降低，而 DCM-RQ 組 (p<0.01) 也 可 顯 著 預 防 GPx 活 性 降 低 ， 但 避 免 GPx 活 性 降 低 之 效 果 與 DCM-RQ 組無顯著差異，至於蛹蟲草內功效成分 CC 與 AS 組則無法有效預 防酒精造成小鼠肝臟內 GPx 酵素活性降低，此外 DOW (p<0.05) 也可有效避免 肝臟內 GPx 酵素活性因酒精導致活性降低。
餵食試驗物質對於酒精性肝損傷小鼠肝臟內 GSH 酵素活性之影響，結果如 圖 4-15 所顯示，EtOH 組相較於 Nor 組 (p<0.001) GSH 酵素含量顯著降低，
可知長期飲用酒精會導致 GSH 酵素被大量消耗，但經過餵食 UPW-CM 之 UCM-RQ 組 (p<0.01) 相較於 EtOH 組 GSH 酵素含量顯著增加，因此得知 UPW-CM 有助於預防酒精大量消耗 GSH，避免氧化壓力累積於肝臟細胞內，此 外 DCM-RQ (p<0.001) 組也可有效提升小鼠肝臟內 GSH 酵素含量，且提升 GSH 酵素含量之效果較優於 UCM-RQ 組，而餵食蛹蟲草內功效成分蟲草素及 腺苷之 CC 與 AS 組雖然可增加 GSH 含量，但僅 AS (p<0.001) 組提升 GSH 酵素含量之效果達顯著差異，而 DOW 對於 GSH 則無法有效提升酵素含 量。
整體結果顯示 DOW-CM 相較於其他試驗物質對於 SOD、CAT、GPx 及 GSH 酵素活性或含量，均具有較佳之提升效果，因此比起其他試驗物質更可有 效預防酒精引起小鼠肝臟內抗氧化能力降低，避免酒精造成肝臟內氧化壓力累積 對肝臟造成氧化損傷。
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圖 4-11 DOW 培養之蛹蟲草發酵產物對小鼠肝臟內 TBARS 之影響
Fig 4-11 The effect of C. militaris-fermented products cultured with DOW on TBARS content of alcohol induced liver injury mice. Data are presented as mean ± SD (n=8).
＃,＃＃,＃＃＃
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH group *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ group.
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圖 4-12 DOW 培養之蛹蟲草發酵產物對小鼠肝臟內 SOD 酵素活性之影響 Fig 4-12 The effect of C. militaris-fermented products cultured with DOW on SOD activity of alcohol induced liver injury mice. Data are presented as mean ± SD (n=8).
＃,＃＃,＃＃＃
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH group *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ group.
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圖 4-13 DOW 培養之蛹蟲草發酵產物對小鼠肝臟內 CAT 酵素活性之影響 Fig 4-13 The effect of C. militaris-fermented products cultured with DOW on CAT activity of alcohol induced liver injury mice. Data are presented as mean ± SD (n=8).
＃,＃＃,＃＃＃
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH group *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ group.
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圖 4-14 DOW 培養之蛹蟲草發酵產物對小鼠肝臟內 GPx 酵素活性之影響 Fig 4-14 The effect of C. militaris-fermented products cultured with DOW on GPx activity of alcohol induced liver injury mice. Data are presented as mean ± SD (n=8).
＃,＃＃,＃＃＃
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH group *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ group.
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圖 4-15 DOW 培養之蛹蟲草發酵產物對小鼠肝臟內 GSH 酵素含量之影響 Fig 4-15 The effect of C. militaris-fermented products cultured with DOW on GSH content of alcohol induced liver injury mice. Data are presented as mean ± SD (n=8).
＃,＃＃,＃＃＃
indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with EtOH group *,**, *** indicated the significant difference (p < 0.05, p < 0.01 and p < 0.001, respectively) as compared with DCM-RQ group.
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九、DOW 發酵之蛹蟲草對酒精性肝損傷模式小鼠肝臟內發炎蛋白之影響 長期飲用酒精目前已知容易導致肝臟發炎 (Louvet and Mathurin., 2015)，本 實驗酒精誘發肝損傷模式小鼠肝臟內發炎相關蛋白質結果顯示， EtOH 組 (p<0.05) 相較於 Nor 組 發炎相關蛋白質 TNF-α 與 NF-κB 均顯著提高 ， COX-2 表 現 量 也 提 高 ， 而 抗 發 炎 相 關 蛋 白 PPAR-γ 表 現 量 則 是 EtOH 組 (p<0.05) 顯著低於 Nor 組。餵食蟲草素的 CC 組 (p<0.001) 小鼠肝臟內 TNF-α、
NF-κB 及 COX-2 等發炎蛋白質表現量均極顯著低於 EtOH 組，且抗發炎相關 蛋白質 PPAR-γ 比起 EtOH 組蛋白質表現量增加，因此可知蟲草素具有降低發 炎相關蛋白及提高抗發炎相關蛋白含量之效果。而餵食腺苷的 AS 組 (p<0.001) 也可顯著降低小鼠肝臟內 NF-κB、TNF-α 及 COX-2 等發炎蛋白含量，以及提 高 PPAR-γ 含量，且 AS 組降低 NF-κB、TNF-α 及 COX-2等發炎蛋白和提高 PPAR-γ 蛋白質含量的效果更是優於 CC 組。UCM-RQ 組的 western blot 結果 顯示小鼠肝臟內 TNF-α (p<0.001)、NF-κB (p<0.001) 及 COX-2 (p<0.01) 蛋白質 含量均顯著低於 EtOH 組，而 PPAR-γ 含量 UCM-RQ 組高於 EtOH 組。此外 DCM-RQ 組也可顯著降低 TNF-α (p<0.001)、NF-κB (p<0.05) 與 COX-2 (p<0.01) 等發炎蛋白含量，以及提高抗發炎蛋白 PPAR-γ 的表現量，但 DCM-RQ 組 (p<0.05) NF-κB 蛋 白 含 量 顯 著 高 於 UCM-RQ 組 ， 可 是 PPAR-γ 含 量 則 DCM-RQ 組高於 UCM-RQ 組。DOW 組 TNF-α (p<0.001)、NF-κB (p<0.05) 及 COX-2 (p<0.001) 含 量 均 顯 著 低 於 EtOH 組 ， 而 DOW 組 (p>0.05) 與 DCM-RQ 組相比 TNF-α、NF-κB 及 COX-2 表現量並無顯著之差異 ，另外 DOW 組抗發炎相關蛋白 PPAR-γ 表現量略高於 EtOH 組。
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圖 4-16 DOW 培養之蛹蟲草對小鼠肝臟內 TNF-α、NF-κB、PPAR-γ 及 COX-2 之影響
Fig 4-16 The effect of C. militaris-fermented products cultured with DOW on liver
Fig 4-16 The effect of C. militaris-fermented products cultured with DOW on liver