第四章 討論
4.8 純化多醣之分子量
以標準品Blue dextran T2000、Dextran T500、Dextran T70、Dextran T40 及 Dextran T10 製作檢量線，分別計算 purified NPP 及 purified NFP 的分子量範圍約為149−607 kDa 與 27−261 kDa (圖 9)。林 (2013)以相同 條件，文旦柚籽純化的多醣分子量範圍約為 176−589 kDa。Zheng 等人 (2016)在黃蜀葵花 (Abelmoschus manihot)純化出一主要多醣 AMPS-a，分 子量為 8.8 kDa。Wang 等人 (2010)純化綠茶花 (Camellia sinensis)多醣 (TFPS1)的分子量為 500 kDa。Hong 等人 (2012)將歐亞璇覆花 (Inula britannica)以 HPLC 測分子量為 3500 Da 與 700 Da。上述結果說明不同 物種多醣的分子量分布都不相同。
Zheng等人 (2014)萃取秋葵 (Abelmoschus esculentus) 花多醣，並以 DE-52 cellulose樹脂及SephacrylTM S-500 膠體分離出一純化多醣，並進 一步利用高壓粒徑排除層析/多角雷射散射 (high pressure size exclusion chromatography/multi-angle laser light scattering, SEC-MALLS) 確認其多 醣的分子量約為 1700 kDa。Tabarsa等人 (2017)將粗萃藥蜀葵(Althaea officinalis)花的多醣以SEC測得的分子量約為 33300 kDa。未來若將 purified NPP及purified NFP經SEC-MALLS分析可獲得更精確的平均分 子量。
4.9
4.10
傅立葉轉換紅外線光譜 (FT-IR)
圖10 為利用FT-IR分析經過DE-52、S-400 層析純化、透析脫鹽後的 乾燥樣品purified NPP及purified NFP之圖譜。多醣的特徵峰在 3600 cm-1–3200 cm-1、3000 cm-1–2800 cm-1、1400 cm-1–1200 cm-1及 1200 cm-1–1000 cm-1 (Wang et al., 2010)。在 2500 cm-1–3600 cm-1有兩個明顯的 波峰，其中3400 cm-1附近的O—H拉伸震動是因為glycopyranose hydroxyl groups的氫鍵結 (Yuen et al., 2009)，2940 cm-1附近為aliphatic的CH震動 (Tabarsa et al., 2017)。在 1736 cm-1、1634 cm-1及1430 cm-1附近強訊號為 酸性多醣，其中 1736 cm-1附近是酯化羧基group (—COOH)及 (—
COOCH3)，1634 cm-1及 1430 cm-1附近為uronic acid的殘基 (—COO–) (Zhao et al., 2010)。Wang等人 (2010)提到在 1643 cm-1與 1416 cm-1附近也 可能是acylamino，推測有蛋白質的存在。Purified NPP在 1736 cm-1有強 訊號為esterified carboxylic groups (—COOR)，往後可經由甲基化測其 CH3判斷是否為 (—COOCH3)或 (—COOH)。Purified NPP在 1429 cm-1及 1332 cm-1有強訊號，可能是C—H與C—O彎曲或拉伸 (Xu et al., 2015)。
在 1250 cm-1附近為S—O震動，說明多醣可能含有硫酸脂 (Wang et al., 2017)。在 950 cm-1–1200 cm-1是碳水化合物中pyranoid ring的指紋帶且可 能為β–型式，因此推測purified NPP及purified NFP皆屬於pyran殘基以β 鍵結的多醣。
蓮花胎座及花梗多醣體對 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 定樣品對細胞是否具有毒性。圖11 顯示，於細胞培養液添加 5–300 μg/mL
的purified NPP 並培養 24 小時，對小鼠纖維母細胞的細胞存活率皆無影 響 (P<0.05)；而添加 purified NFP 培養 24 小時，發現對細胞是有毒性的，
且在濃度5 μg/mL 以上就有毒性。圖 12 A 顯示健康且未做添加處理的小 鼠纖維母細胞外觀型態，可看出細長型外觀，兩端如手臂抓緊培養盤，
且覆蓋性高，像表皮層層保護般。添加100 μg/mL purified NPP 培養 24 小時後的細胞型態並無改變 (圖 12 B)；而添加 purified NFP 培養 24 小 時後的細胞型態雖無變化，但數量有減少現象 (圖 12 C)。
纖維母細胞 (fibroblasts)是真皮層 (dermis)的主要細胞 (張，2014)，
因此可初步認為NPP 使用於化妝原料在濃度低於 300 μg/mL 對皮膚沒有 毒性。
第五章 未來研究方向
根據以上試驗結果，未來方向可分成學術及應用研究
在學術研究方面：
1.
使用統計實驗設計方法，藉由改變溫度、時間、固/液比等參數，找尋NPP 及 NFP 的最佳萃取條件。
2.
利用high pressure size exclusion chromatography (HPSEC)更精確分 析purified NPP 及 purified NFP 的分子量。3.
使用scanning electron microscope (SEM)掃描式電子顯微鏡觀察 NPP 及NFP 與 purified NPP 及 purified NFP 外觀的差異。4.
使用 thermogravimetric analyzer (TGA)熱重量分析觀察 NPP 及 NFP 與 purified NPP 及 purified NFP 的熱崩解情況。在應用研究方面：
1.
NPP 具有良好的保水能力及抑制酪胺酸酶的效果，期待做為添加化 妝品的可能性。且具有高黏稠度的特性，可添加甚至取代食品增稠 劑等方面的應用。2.
試驗purified NPP 及 purified NFP 是否具有抗醣化、抗發炎作用或抗 腫瘤活性。3.
近年來，天然多醣的化學修飾已引起人們極大的關注，可以用於修飾 結 構 以 達 到 特 定 的 目 的 ， 並 產 生 新 的 生 物 活 性 (Jiang et al., 2015)。因此，可嘗試化學修飾蓮花多醣。
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