粗多糖提取及纯化方法

粗多糖水提醇沉提取方法

第一种方法：

（1）玉米须原料用80%乙醇在78℃提取三次，去除色素、单糖和脂溶成分。过滤，合并滤渣，使乙醇挥发干净，粉碎滤渣。

用100℃热水浸提干燥玉米须一小时，料液比1：15，提取三次。合并提取液、过滤和浓缩，利用三倍体积80%乙醇醇沉。

（2）离心收集醇沉物(3000 ×g，10min)，然后用水溶解，并利用Sevag 方法去除游离蛋白质。

（3）脱去蛋白质的溶液再次用80%乙醇醇沉。收集沉淀，先后用无水乙醇和丙酮溶液冲洗三次。最后冷冻干燥，获得玉米须粗多糖。

Hot water extraction of crude polysaccharides

All the corn silk raw materials were immersed in 80% (v/v) ethanol at 78℃ for three times to remove colored materials, monosaccharide and liposoluble constituents. The organic solvent was volatilized and th

e pretreated corn silk was obtained to crush. Dried ground corn silk (10

0 g) was extracted with water at 100℃(1:15 (w/v), 1 h, 3 times). The ext raction solutions were combined, filtered and concentrated, precipitate d by the addition of anhydrous ethanol to a final concentration of 80% (v/v). Precipitates were collected by centrifugation (3000 × g, 10 min), then dissolved with water, and subjected to the Savage method (chlorofor m: butyl alcohol, 4:1) to remove free proteins. The deproteinized solutio n was re-precipitated in 80% (v/v) ethanol. The precipitates were collect ed and successively washed with anhydrous ethanol and acetone. After freezing dried, the hot water extraction corn silk polysaccharides (PS) we re obtained.

第二种方法

Preparation of ILPSThe preparation of crude ILPS was carried out according to thereported method with some modifications (Xu e t al., 2012). Briefly,the powder of I. latifolia Thunb was pre-extract ed two times with85% aqueous ethanol solution (v/v) in a ratio of 1:15 (materialto ethanol solution, g/mL) at 80◦C for 1 h each, and the super-natant (containing colored materials, oligosaccharides and s mallmolecule compounds) was removed. The resulting residues were extracted three times with distilled water in a ratio of materialto wa ter 1:20 (g/mL) at 90◦C for 3 h each, and the extracts werecentrifu ged at 5000 rpm for 15 min. The supernatants were com-bined and concentrated by a rotary evaporator to a proper volume.The resulti ng concentrate was mixed with three times volume ofabsolute ethan ol, stirred vigorously and kept overnight at 4◦C. Theprecipitates wer e then collected by centrifugation at 5000 rpm for15 min, dissolved in distilled water, dialyzed 透析against distilled waterand lyophiliz ed, affording the crude ILPS.

纯化：

The crude ILPS was purified by chromatography of DEAEcellu lose-52 according to the reported method (Qiao et al., 2009a;Ye, W ang, Zhou, Liu, & Zeng, 2008). Briefly, 150 mg of crude ILPS wa s dissolved in 7.5 mL deionized water, and the solution was filtere d through a 0.45 _m membrane filter. The resulting ILPS solution wasthen loaded onto a column (2.6 cm ×30 cm) of DEAE cellu lose-52,and the column was stepwise eluted with deionized water, 0. 1,0.3 and 0.5 M sodium chloride (NaCl) solution at a flow rate of1.

0 mL/min. The eluate was collected automatically (10 mL/tube)and the carbohydrate in each tube was determined by the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith,1956). Th