第四章 討論
4.4. 柚籽多醣體的特性
4.4.4 柚籽多醣體對 NIH-3T3 細胞的毒性分析
細胞死亡主要為細胞壞死 (necrosis)及細胞凋亡 (apoptosis)兩種途 徑。細胞壞死是細胞受到外在因子 (例如輻射、紫外線等)或毒素所 引起，在光學顯微鏡下可觀察到細胞腫大、破裂，且胞內物質流出 (Majno and Joris, 1995; Broker et al., 2005 )。由活細胞數目及細胞外觀 型態，可判定樣品對細胞的毒性。圖 3-8 顯示，於細胞培養液添加不 同濃度的 WE 與 Fraction B，對小鼠纖維母細胞的細胞存活率皆無影 響。圖 3-9 顯示，添加 WE 與 Fraction B 培養 24 小時後的細胞型態，
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添加前後皆無改變。綜合上述結果，WE 與 Fraction B 對小鼠纖維母 細胞不具細胞毒性。纖維母細胞 (fibroblasts)是真皮層 (dermis)的主 要細胞 (光井武夫，2005)，因此可初步認為柚籽黏質用於化妝原料 對皮膚沒有刺激性。
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第五章 未來研究方向
根據以上試驗結果，未來將有以下幾項發展方向：
1. 柚籽可做為一萃取多醣體的理想原料。但由於柚籽於室溫的萃取 率偏低，將繼續試驗最適萃取條件。
2. 利用氣相層析質譜與核磁共振儀解析Fraction B的未知醣醛酸及 醣苷鍵結，以確認Fraction B的果膠類型。
3. 分析Fraction A的單醣組成、醣苷鍵結及分子量，有助於了解柚籽 黏質的高黏度特性及凝膠機制。
4. 試驗溫度、pH值及陽離子濃度等條件對柚籽黏質的黏度之影響。
5. 分析水萃及有機溶劑萃取的柚籽黏質內，是否具有多元酚含量。
6. 解析柚籽黏質內的SOD活性來源物質。
7. 改良螯合亞鐵離子能力的試驗方法。可加入緩衝溶液使反應液為 中性，降低對實驗結果的影響。
8. 試驗柚籽黏質是否具有抗發炎作用。
9. 試驗柚籽黏質是否具有抗腫瘤活性。
10. 柚籽黏質有良好的水合能力。可利用角質含水測定儀(corneometer) 及穿皮水份流失儀(transepidermal water loss meter, TEWL)檢測柚 籽黏質對皮膚含水量的影響。
11. 初步認為柚籽黏質對於皮膚沒有刺激性。可利用皮膚測試貼布 (skin patch test)，進行柚籽黏質對人體皮膚的過敏性評估。
12. 將柚籽黏質實際應用於食品、化妝品及藥物的添加劑。
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表 3-1. 室溫水萃柚籽表面黏質物 (WE)的產率
Extract weight/seed dry weight (%)
WE 2.9 ± 0.0
WE 的產率是 WE 乾重相對於柚籽乾重之比率，數據為平均值± SEM (n=3)。
表 3-2. 以 DE-52 樹脂分離 WE 得到兩 fraction 的回收率
各 fraction 的回收率是回收的重量相對於所注入的 WE 乾重，Fraction A 是未吸 附的多醣，Fraction B 是 0.100.18 M NaCl 流洗出的多醣。數據為平均值± SEM (n=3)。
表 3-3. WE 與 Fraction B 的總醣、醣醛酸及蛋白質含量比率
數據為平均值± SEM (n=3)。
Fraction weight/WE weight (%)
Fraction A 8.3 ± 2.1
Fraction B 59.7 ± 0.6
Composition (%) WE Fraction B
Total sugar (as glucose equivalent) 42.6 ± 10.6 16.3 ± 3.7 Uronic acid (as galacturonic acid equivalent) 38.7 ± 3.6 53.4 ± 3.8 Protein (as albumin equivalent) 0.1 ± 0.0 0.2 ± 0.1
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表 3-4. Fraction B 內中性單醣的相對比例
Relative proportions of neutral monosaccharides in Fraction B
Rhamnose 5
Arabinose 5
Galactose 3
Glucose 2
Mannose or Xylose 4
Fraction B 以 TFA 水解後，利用離子液相層析系統分析 Fraction B 的中性單醣之 相對比例。數據為三批的平均值。
表 3-5. Fraction B 的甲基化程度
Degree of methylation (%)
Fraction B 5.7 ± 1.3
樣品的甲基含量對醣醛酸含量之比率，數據為平均值± SEM (n=3)。
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表 3-6. WE、Fraction B 與 Gluten 的水合能力
Water hydration capacity (mL/g DW)
WE 9.4 ± 0.3
Fraction B 15.2 ± 1.5
Gluten from wheat 1.3 ± 0.0
每克樣品乾重可水合的毫升數。數據為平均值± SEM (n=3)。
表 3-7. WE與Fraction B的Superoxide dismutase活性
SOD (units / g DW)
WE 7300 ± 700
Fraction B 3600 ± 300
EGCG 73300 ± 2500
每克樣品乾重所含的SOD活性單位。數據為平均值± SEM (n=3)。
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圖 3-2. 以液相層析系統分析 Fraction B 的醣類成份層析圖
Fraction B 以 TFA 水解後，以液相層析系統 (Carbo PA10, Dionex)分析中性單醣 成份，圖中 y 軸為移動相中導電度的變化，x 軸則為分離物質的滯留時間。(A) peak 1 為 rhamnose，peak 2 為 arabinose，peak 3 為 galactose，peak 4 為 glucose，peak 5 為 mannose 或 xylose。(B) Fraction B 以 H2SO4水解後，以液相層析系統 (IonPac AS11, Dionex)分析醣醛酸成份，peak 2 為 Galacturonic acid，peak 3 為 Glucuronic acid，peak 1、4、5 則為未知物。
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Absorbance
0.0 0.4 0.8
1.2 T 40
T 2000
T 500
T 70 (A)
Elution Volume (mL)
80 100 120 140
Absorbance
0.08 0.12 0.16
(B)
圖 3-3. 標準品 (A)與 Fraction B (B)在 Sephacryl® S-400 的分子篩層析圖 在 Sephacryl® S-400 以 0.2 M NaCl buffer 流洗標準品Blue dextran T2000、Dextran T500、Dextran T500、Dextran T40 與 Fraction B。(A) 以 660 nm 吸光值檢測 Blue dextran T2000 的溶離體積；依照總醣含量測定，以 490 nm 吸光值檢測其他標準 品的溶離體積。(B) 依照醣醛酸含量測定，以 520 nm 吸光值檢測 Fraction B 的 溶離體積。
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Molecular Weight (kDa)
100 1000
K a v
0.00 0.25 0.50
T 40
T 70
T 500
T 2000 Fraction B
Fraction B
圖 3-4. 決定 Fraction B 的分子量
以標準品Blue dextran T2000、Dextran T500、Dextran T70 及 Dextran T40 在
Sephacryl® S-400 的 Kav 對應底數為 10 的對數分子量，製作 Kav 檢量線，並內
插計算 Fraction B 的 Kav，得知 Fraction B 的分子量。
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Concentration (%, w/v)
0.00 0.25 0.50 0.75 1.00
Viscosity (cP)
0 2000 4000 6000
WE
Acacia gum
圖 3-5. 不同濃度的 WE 與阿拉伯膠水溶液於 27℃的黏度變化 數據為平均值± SEM (n=3)。
58 Concentration (mg/mL)
0 1 2 3 4
Scavenging (%)
0 40 80
WE Fraction B Ascorbic acid (A)
0.00 0.25 0.50 0.75 1.00
Ascorbic acid (B)
圖 3-6. 不同濃度的 WE、Fraction B 及 ascorbic acid 對清除 DPPH 自由基活性 之影響
(A) WE、Fraction B 以及 ascorbic acid 對 DPPH 自由基的清除能力。(B) 低 ascorbic acid 濃度對 DPPH 自由基的清除能力。數據為平均值± SEM (n=3)。
59 Concentration (mg/mL)
0 1 2 3 4
Chelating Effects (%)
0 50
100 WE
Fraction B EDTA
0.00 0.25 0.50 0.75 1.00
EDTA
(A) (B)
圖 3-7. 不同濃度的 WE、Fraction B 及 EDTA 對螯合亞鐵離子活性之影響 (A) WE、Fraction B 以及 EDTA 對亞鐵離子的螯合能力。(B) 低 EDTA 濃度對亞 鐵離子的螯合能力。數據為平均值± SEM (n=3)。
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圖 3-9. 以倒立式相位差顯微鏡觀察小鼠纖維母細胞經 WE 與 Fraction B 處理 24 小時後之細胞型態變化
(A) 未添加 WE 的細胞外觀。(B) 未添加 Fraction B 的細胞外觀。(C) 含 10 μg/mL WE 的細胞外觀。(D) 含 10 μg/mL Fraction B 的細胞外觀。
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