壯瑤藥材馬蹄蕨多糖的提取工藝及其3種單糖的含量測定方法研究

　　中圖分類號 R914.1 文獻標(biāo)志碼 A 文章編號 1001-0408（2018）19-2667-04
　　DOI 10.6039/j.issn.1001-0408.2018.19.17
　　摘 要 目的：對壯瑤藥材馬蹄蕨的多糖成分進行提取分離純化，并建立同時測定其中鼠李糖、果糖和D-無水葡萄糖3種單糖含量的方法。方法：優(yōu)化水提醇沉法提取馬蹄蕨多糖，比較不同型號大孔樹脂除蛋白效果，并考察DEAE-52纖維素柱純化所用不同洗脫液。采用高效液相色譜-蒸發(fā)光散射法進行含量測定。色譜柱為ZORBAX NH2，流動相為乙腈-水（72 ∶ 28，V/V），流速為0.8 mL/min，柱溫為40 ℃，進樣量為10 μL。漂移管溫度為50 ℃，載氣流速為2.0 L/min，增益為1。結(jié)果：采用水提75%乙醇沉淀提取馬蹄蕨粗多糖，D101大孔樹脂除蛋白質(zhì)，DEAE-52纖維素柱純化，并依次用蒸餾水、0.2 mol/L NaCl、0.2 mol/L NaOH溶液進行洗脫，冷凍干燥，即得馬蹄蕨多糖。鼠李糖、果糖和D-無水葡萄糖檢測質(zhì)量濃度線性范圍分別為49.10～982.02、54.47～1 089.42、62.15～1 242.96 μg/mL（r≥0.999 6）；檢測限分別為7.4、3.2、4.4 μg/mL，定量限分別為24.4、10.6、14.5 μg/mL；精密度、穩(wěn)定性（15 h）試驗的RSD均<2%（n=6）；重復(fù)性試驗的RSD<3%（n=6）；平均加樣回收率分別為96.91%、97.43%、99.46%，RSD分別為1.3%、1.7%、2.2%（n=6）。結(jié)論：建立的多糖提取分離工藝簡單，易操作；高效液相色譜-蒸發(fā)光散射法操作簡單，結(jié)果準(zhǔn)確度高，可用于馬蹄蕨多糖的提取純化及其中3種單糖的定量分析。
　　關(guān)鍵詞 馬蹄蕨；壯瑤藥材；多糖；提取分離；含量測定；高效液相色譜-蒸發(fā)光散射法
　　ABSTRACT OBJECTIVE： To extract， separate and purify the polysaccharides from Zhuang and Yao medicine Angiopteris fokiensis， and to establish a method for simultaneous determination of isodulcite， fructose and D-anhydrous glucose in 3 kinds of monosaccharide. METHODS： Optimization of water extraction and alcohol precipitation for extracting A. fokiensis polysaccharide，compare the effect of different types of macroporous resin on protein removal， and investigate the different eluents used in the purification of DEAE-52 cellulose column. HPLC-ELSD method was used. The determination was performed on ZORBAX NH2 column with mobile phase consisted of acetonitrile-water （72 ∶ 28， V/V） at the flow rate of 0.8 mL/min. The column temperature was 40 ℃ and sample size was 10 μL. The drift tube temperature was 50 ℃， the carrier gas velocity was 2.0 L/min， and the gain was 1. RESULTS： Extraction of crude polysaccharides from the horseshoe fern by water extraction with 75% ethanol， D101 macroporous resin in addition to protein，DEAE-52 cellulose column for purification， and eluted sequentially with distilled water， 0.2 mol/L NaCl， and 0.2 mol/L NaOH solution， freeze-dried to obtain the A. fokiensis polysaccharide. The linear range of isodulcite， fructose and D-anhydrous glucose were 49.10-982.02 μg/mL， 54.47-1 089.42 μg/mL， 62.15-1 242.96 μg/mL （r≥0.999 6）. The detection limits were 7.4， 3.2 and 4.4 μg/mL， and the quantitation limits were 24.4， 10.6， 14.5 μg/mL. RSDs of precision and stability （15 h） test results were all lower than 2% （n=6）. RSDs of reproducibility tests were all lower than 3% （n=6）. The average recoveries were 96.91%， 97.43% and 99.46%； RSD were 1.3%， 1.7%， 2.2%（n=6）， respectively. CONCLUSIONS： The extraction and isolation technology of polysaccharides are simple and simple to operate. The HPLC-ELSD method is simple， accurate and suitable for the separation and purification of polysaccharides from A. fokiensis and quantitative analysis of 3 kinds of monosaccharides.

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