CN117343207A - 一种新疆大果沙枣纯化多糖的制备方法及应用 - Google Patents
一种新疆大果沙枣纯化多糖的制备方法及应用 Download PDFInfo
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Abstract
本发明涉及新疆大果沙枣多糖及其制备方法和用途，属于药物和保健品领域。本发明采用新疆喀什地区的大果沙枣，首次制备出一种水溶性新疆大果沙枣多糖纯品EMP‑1，结构分析表明所得大果沙枣多糖为酸性多糖，EMP‑1的相对分子量为13.80kDa，主要由不同比例的鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖和半乳糖醛酸组成，摩尔比为12.7％：16.1％：38.7％：11.8％：11.2％：9.6％。以过量酒精损伤正常人胃黏膜上皮细胞GES‑1为模型进行免疫活性评价，结果表明EMP‑1可显著缓解机体的氧化应激状态。具有较好的免疫调节活性，为新疆大果沙枣多糖在食品和医药领域的开发应用提供理论依据，有利于实现新疆大果沙枣资源的高值化利用。
Description
技术领域
本发明涉及一种多糖的制备方法及应用，尤其是一种以新疆大果沙枣为原料提取纯化的大果沙枣多糖的制备方法及应用。
背景技术
随着经济状况的改善和酒精产量的增加，饮酒的成年人比例有所上升。因此，与饮酒有关的疾病的患病率也在增加。摄入过多酒精会对胃黏膜造成直接损伤，通常会引起消化道溃疡、急性胃黏膜出血病变以及急性糜烂性胃炎等疾病。治疗胃损伤的主要手段通俗点来讲就是增加胃内保护因子的成分或者降低侵略者在胃内的影响力。通常使用的药物有许多局限性（高复发率、副作用、后遗效应、药物依赖性和药物相互作用等）增加了对新治疗选择的需求。在这种情况下，从天然物质或功能性食品中提取的生物活性物质已成为有效治疗急性胃溃疡的新方法。
天然来源的植物多糖作为一种来源丰富、安全、疗效高、相对无毒、生物相容性好、价值可观的活性成分，因其优异的理化性质和多种药理活性，已被发现具有多种生物活性，包括清除自由基、抑制脂质氧化、促进自然杀伤细胞(NK)细胞毒性、激活巨噬细胞和白细胞介素等潜在的抗氧化和免疫调节活性以及抗肿瘤和抗凝血等。在药物副作用严重的背景下，天然多糖在预防和治疗疾病方面将得到验证，并展现其成本低、低毒高效的优势。可用于开发新型慢性疾病药物和治疗方法。更重要的是，这些聚合物在化妆品、食品和药物配方开发等领域也得到了广泛的应用。
新疆大果沙枣富含糖类、类黄酮、脂肪酸、萜类化合物、蛋白质和维生素等营养物质，还包括钾、镁、钠等有益的无机元素。其作为维吾尔族传统药材，被广泛用于放松肌肉、缓解疼痛、治疗炎症、阿米巴痢疾呕吐、黄疸、哮喘、退烧和治疗溃疡。
目前，所报道的新疆沙枣多糖仅限于粗多糖（混合多糖），而非新疆大果沙枣多糖纯品（单一多糖），即未进行除杂、分离、纯化等步骤，粗多糖含有大量的蛋白分子、色素、无机盐以及其他小分子杂质，产品纯度低，结构复杂且未知等因素严重影响其生物学活性的研究进程。
发明内容
本方案是为了解决现有技术所存在的上述技术问题，提供一种新疆大果沙枣多糖的制备方法及应用。
为解决上述问题，本发明采用的技术方案为：一种新疆大果沙枣多糖EMP-1，其相对分子量为13.80 kDa，主要由不同比例的鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖和半乳糖醛酸组成，摩尔比为12.7%：16.1%：38.7%：11.8%：11.2%：9.6%。
一种上述新疆大果沙枣多糖的制备方法，其特征在于依次按照如下步骤进行:
a . 原料粉碎：将新鲜大果沙枣经除杂、清洗、于太阳下晾晒一天除去大部分水分，后置于50 ℃烘箱干燥过夜， 烘干至重量恒定且水分含量5 %以下。人工将果肉和果核分离，用粉碎机粉碎后，过100目筛将其转化成细粉；
b. 热水提取：取大果沙枣细粉与去离子水(1:30 g/mL)混合，90 ℃搅拌提取3小时，重复提取三次。抽滤后合并提取液，通过真空旋转蒸发程序将提取液浓缩，得浓缩液；
c . 醇沉：在浓缩液中加入四倍体积的95%乙醇沉淀24 h。沉淀产物用丙酮和乙醚洗涤三次，经冷冻干燥得到大果沙枣粗多糖；
d . 脱色素：采用大孔树脂D-101脱色素。先用无水乙醇将大孔树脂浸泡24 h备用，采用高60 cm，内直径3 cm规格的层析柱，装柱时采用湿法上柱，用去离子水反复洗脱大孔树脂直至滴出液体无浑浊无醇味，将沙枣粗多糖溶液缓慢倒入层析柱中进行洗脱去除色素。真空旋蒸至适量体积，经冷冻干燥后得到脱色素粗多糖；
e . 脱蛋白：采用酶法脱蛋白。简而言之，将大果沙枣粗多糖溶解于去离子水中，55 ℃与木瓜蛋白酶反应2 h，持续搅拌。100 ℃沸水浴10 min后离心去除变性蛋白。将上清液浓缩后冷冻干燥，得到脱蛋白多糖；
f . 离子交换柱层析：将脱色和脱蛋白的沙枣多糖溶于超纯水中，载于DEAE-52色谱柱(3 cm × 60 cm)上。用超纯水平衡2 h后，用0.3 mol/L的NaCl溶液梯度洗脱，流速为2mL/min，每管10 mL，苯酚-硫酸法在线监测。根据洗脱曲线自动采集组分。真空旋蒸浓缩，透析脱盐(3500 Da)后冷冻干燥，最终得到多糖粗品；
g. 凝胶过滤柱层析：Sephadex G-100凝胶色谱柱以双蒸水洗脱多糖粗品EMP-1，浓缩冷冻干燥后获得一种多糖纯品，命名为大果沙枣纯化多糖EMP-1。
一种上述的新疆大果沙枣多糖制备激活免疫细胞药物中的应用。
本发明是以新疆大果沙枣为原料，采取水提醇沉法获得粗多糖，再对粗多糖进行脱色素、脱蛋白和除盐处理，最后利用离子交换柱层析和凝胶过滤柱层析对粗多糖进行分离纯化，首次制备出一种水溶性大果沙枣多糖纯品EMP-1，其相对分子量为13.80 kDa，主要由不同比例的鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖和半乳糖醛酸组成，摩尔比为12.7%：16.1%：38.7%：11.8%：11.2%：9.6%。所制备的大果沙枣多糖EMP-1具备免疫调节活性，可以显著降低酒精诱导的GES-1损伤细胞中活性氧ROS的活化和提高抗氧化酶的活性（超氧化物歧化酶SOD、过氧化氢酶CAT和谷胱甘肽过氧化物酶GSH-Px），降低髓过氧化物酶MPO的外溢、丙二醛MDA脂质过氧化，缓解机体的氧化应激状态。可应用于饲料添加剂、食品、化妆品、保健品及药品等领域。
附图说明
图1是新疆大果沙枣多糖EMP-1的制备工艺流程图。
图2是新疆大果沙枣多糖EMP-1的DEAE-52纤维素阴离子交换柱层析洗脱图。
图3是新疆大果沙枣多糖EMP-1的Sephadex G-100凝胶色谱柱层析洗脱图。
图4是新疆大果沙枣多糖EMP-1的分子量图。
图5是新疆大果沙枣多糖EMP-1的单糖组成与标准品对比图。
图6是新疆大果沙枣多糖EMP-1的紫外吸收光谱图。
图7是新疆大果沙枣多糖EMP-1的红外吸收光谱图。
图8是新疆大果沙枣多糖EMP-1的扫描电镜图。
图9是新疆大果沙枣多糖EMP-1的热稳定性图。
图10是新疆大果沙枣多糖EMP-1的刚果红实验图。
图11是新疆大果沙枣多糖EMP-1的体外细胞增殖图。
图12是酒精损伤GES-1细胞后EMP-1的治疗作用图。
图13是新疆大果沙枣多糖EMP-1的活性氧ROS作用图。
图14是新疆大果沙枣多糖EMP-1的超氧化物歧化酶SOD作用图。
图15是新疆大果沙枣多糖EMP-1的过氧化氢酶CAT作用图。
图16是新疆大果沙枣多糖EMP-1的谷胱甘肽过氧化物酶GSH-Px作用图。
图17是新疆大果沙枣多糖EMP-1的丙二醛MDA作用图。
图18是新疆大果沙枣多糖EMP-1的髓过氧化物酶MPO作用图。
其中各组分别为：正常组(Control)、模型组(Ethonal)、实施例组1(5 ug/mL)、实施例组2(10 ug/mL)和实施例组3(20 ug/mL)。数据以均值±标准差表示( n＝6)。组间显著差异（p<0.05）用不同的字母（a-f）表示。
具体实施方式
下面将结合具体实施例，对本发明的技术方案进行清楚、完整地描述，但是，所描述的实施例仅为本发明一部分实施例，并不是全部的实施例。本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：如图1所示，一种具有免疫活性作用的新疆大果沙枣均一性多糖制备方法，包括以下步骤：
a . 原料粉碎：将新鲜大果沙枣经除杂、清洗、于太阳下晾晒一天除去大部分水分，后置于50 ℃烘箱干燥过夜， 烘干至重量恒定且水分含量5 %以下。人工将果肉和果核分离，用粉碎机粉碎后，过100目筛将其转化成细粉；
b. 热水提取：取大果沙枣细粉与去离子水(1:30 g/mL)混合，90◦C搅拌提取3小时，重复提取三次。抽滤后合并提取液，通过真空旋转蒸发程序将提取液浓缩，得浓缩液；
c . 醇沉：在浓缩液中加入四倍体积的95%乙醇沉淀24 h。沉淀产物用丙酮和乙醚洗涤三次，经冷冻干燥得到大果沙枣粗多糖；
d . 脱色素：采用大孔树脂D-101脱色素。先用无水乙醇将大孔树脂浸泡24 h备用，采用高60 cm，内直径3 cm规格的层析柱，装柱时采用湿法上柱，用去离子水反复洗脱大孔树脂直至滴出液体无浑浊无醇味，将沙枣粗多糖溶液缓慢倒入层析柱中进行洗脱去除色素。真空旋蒸至适量体积，经冷冻干燥后得到脱色素粗多糖；
e . 脱蛋白：采用酶法脱蛋白。将大果沙枣粗多糖溶解于去离子水中，55 ℃与木瓜蛋白酶反应2 h，持续搅拌。100 ℃沸水浴10 min后离心去除变性蛋白。将上清液浓缩后冷冻干燥，得到脱蛋白多糖；
实施例2：将脱色和脱蛋白的沙枣多糖溶于超纯水中，载于DEAE-52色谱柱(3 cm× 60 cm)上。用超纯水平衡2 h后，用0.3 mol/L的NaCl溶液梯度洗脱，流速为2 mL/min，每管10 mL，苯酚-硫酸法在线监测。根据洗脱曲线自动采集组分。真空旋蒸浓缩，透析脱盐(3500 Da)后冷冻干燥，最终得到多糖粗品。
实施例3：使用Sephadex G-100凝胶色谱柱以双蒸水洗脱多糖粗品EMP-1，浓缩冷冻干燥后获得一种多糖纯品，命名为大果沙枣纯化多糖EMP-1。
实施例4：采用高效凝胶渗透色谱HPGPC法测定所得的新疆大果沙枣多糖EMP-1的均一性和相对分子量。测试条件：通过串联连接的BRT105-104-102色谱柱( × 300 mm，柱温40 ℃)串联的高效凝胶渗透色谱进行检测，并通过ShodexRI-502检测器检测。测试结果参见图4，经检测，所得的新疆大果沙枣多糖EMP-1为均一多糖，其相对分子质量为13.80kDa。
实施例5：采用离子色谱（IC）分析EMP-1的单糖组成。简而言之，将 2 mL 的 3 MTFA 添加到 5 mg 多糖样品中;然后将混合物用N2并在120 °C下水解3小时。准确吸取酸水解溶液转移至1.5 mL Eppendorf （EP） 管中氮吹吹干，加入5ml水涡旋混匀，吸取50 uL加入950 uL去离子水，12000 rpm离心5 min。取上清液（5 μL）使用离子色谱仪（ICS20）和电化学检测器的DionexCarbopac™ PA3色谱柱（150 × 40 mm，柱温5000 °C）进样。并以超纯水、15 mM氢氧化钠、15 mM氢氧化钠和100 mM乙酸钠的混合物以0.3 mL/min的速度作为流动相。将样品溶液（5 μL）进样，并以超纯水、250 mM氢氧化钠、50 mM氢氧化钠和500 mM乙酸钠的混合物以0.3 mL/min的速度作为流动相运行。岩藻糖、鼠李糖、阿拉伯糖、盐酸氨基葡萄糖、盐酸氨基半乳糖、半乳糖、葡萄糖、N-乙酰-D氨基葡萄糖、木糖、甘露糖、果糖、核糖、葡萄糖醛酸、半乳糖醛酸甘露糖醛酸和古罗糖醛酸作为单糖标准品使用相同的程序进行测定。结果表明EMP-1是一种酸性果胶杂多糖，主要由鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖和半乳糖醛酸组成，摩尔比为12.7%：16.1%：38.7%：11.8%：11.2%：9.6%。
实施例6：采用UV-2600分光光度计在700 ~ 190 nm范围内测定EMP-1 (0.5 mg/mL)溶液的紫外可见(UV/Vis)吸光度。结果表明由于蛋白质在280 nm处有特征吸收，因此可以通过紫外扫描初步判定是否含有蛋白质。紫外扫描显示在280 nm处几乎没有吸收，再次验证了EMP-1的纯度和均一性。
实施例7：将适量干燥的EMP-1与光谱级KBr粉末混合，然后将混合物压成薄片，用FTIR在4000-500 cm−1范围内扫描。结果表明在3365 cm-1处有一个宽阔而强烈的峰，归因于多糖网络中分子间和分子内氢键相互作用引起的O-H的伸缩振动，这是糖类的特征峰，所有多糖都含有。在2929 cm-1处有一个窄的吸收峰，可能归属于醚（甲基或乙酰基）的C-H伸缩振动。在1735 m-1处有一个吸收峰，可能归属于C=O伸缩振动。在1615 m-1和1421 cm-1处有吸收峰，可能归属于不对称和对称C-O伸缩振动。在1249 cm-1处有吸收峰，可能归属于O-H变角振动。在1151 cm-1处有吸收峰，可能归属于C-O-C基团的振动和糖苷桥重叠的环振动。在1076cm-1和1037 cm-1处有吸收峰，可能归属于吡喃环的非对称环伸缩振动。在892 cm-1处有吸收峰，可能归属于吡喃环的β-端基差向异构的C-H变角振动。
实施例8：采用扫描电镜(SEM, Nova NanoSEM 450, 美国)观察纯化多糖EMP-1的表面微观结构。简而言之，取约5 mg干燥后样本，粘附于含有双面粘合剂的导电碳膜上，置于离子溅射仪样品舱中，进行喷金40 s左右，确保每个部分都包金。样品取出后，置入扫描电镜观察室，加速电压为3 KV，在不同倍率(500-20000倍)下观察。如图8所示，样品在500，1000倍数下可见样品呈现分散层片状和聚集的球形状碎片，球形聚集体表面呈现网状，有许多密集小孔堆积在一块，分散性良好，根据多糖分散指数得出这种现象可能是由于大分子之间的强烈吸引力。放大至5000，10000倍数下，可见不规则层片状，分子排列密集，表面光滑。在15000倍及20000倍数下，表面被放大，可见部分鱼皮状表面和串珠状结构，并可见细小的断裂层片贴附于较大的层片表面，孔隙明显较少。这可能是由于EMP-1主要以具有强分子间交联的聚集状态存在。
实施例9：使用STA 449F3热重分析仪进行了一些修改。将质量为5.0 mg的EMP-1样品以10 °C/min的恒定速率从50 °C加热到750 °C，将观测数据绘制成热重（TG）和推导热重（DTG）曲线，以反映EMP-1热转化引起的物理化学变化。大果沙枣多糖EMP-1组分的TGA和DTG曲线如图9所示，在30 ℃ ~ 700 ℃范围内，EMP-1组分的质量损失曲线根据温度可以分为三个阶段。热降解的第一阶段是样品水分的流失。EMP-1在197 ℃ 下失重7.813%。EMP-1的失重与这一阶段沙枣多糖中游离水或结合水的损失有关。第二阶段的热降解发生在200 ℃- 500 ℃，此温度区间的失重过程快速，且失重率比重大，说明EMP-1在这个温度范围内发生了剧烈的分解反应，可能是这由于热不稳定官能团的降解。TGA结果显示，EMP-1在第二阶段失重约63%。最后，当温度超过500 ℃ 时，EMP-1重量保持稳定，基本为矿物质的固体残渣。
实施例10：采用刚果红法，建立了新疆大果沙枣多糖EMP-1馏分的构象。简单地说，3.0 mL刚果红溶液(80 μmol/L)与3.0 mL多糖溶液(1 mg/mL)混合。在初始溶液中加入NaOH溶液(1 mol/L)制备以下每个浓度:0、0.1、0.2、0.3、0.4和0.5 mol/L。然后用紫外可见分光光度计测定溶液的最大吸收波长在300 ~ 700 nm之间。研究表明，具有三螺旋结构的多糖样品在弱碱性条件下可以与刚果红形成配合物。与刚果红相比，它的紫外吸收会发生红移(向波长增大的方向移动)。但随着碱性增大，强碱会破坏生物聚合物的三螺旋构象，分子之间的氢键会断裂，三螺旋结构分解为单链，不能与刚果红形成配合物，导致λmax迅速下降。如图10所示，在NaOH浓度小于0.1 mol/L时，最大吸收峰与空白组相比发生红移，说明EMP-1组分中存在复杂的三螺旋结构，在弱碱情况下与刚果红络合发生生红移，当NaOH浓度大于0.1 mol/L时，最大吸收波长开始表现出下降趋势，表明三螺旋结构解体，变成没有规则的自由卷曲状态。这些结果表明EMP-1具有复杂的三螺旋结构，可能具有良好的生物学活性。
实施例11：人胃黏膜上皮细胞GES-1用于体外研究高剂量乙醇对GES-1细胞的影响。将细胞放在含有10%的胎牛血清(FBS),，1%的青霉素-链霉素和89%的RPMI 1640培养基的中培养。然后在37℃、5% CO2培养箱中孵育。为了确定不同浓度的EMP-1对细胞增殖的影响，取对数生长期状态良好的细胞培养24小时后弃去废液，将浓度为0、5、10、20、40、80、160、320、640和1280 μg/mL的EMP-1加入培养基中。空白对照仅添加RPMI 1640培养基。培养24小时后，弃去上清液。通过cck8试剂盒测量EMP-1干预后的细胞增殖率。简而言之，每孔加入10 ul的CCK8溶液，继续在没有光照的情况下孵育1 h。使用酶标仪记录450 nm处的吸光度值，计算细胞增殖率。细胞增殖实验结果表明，640 μg/mL的GES-1细胞活力>90%，表明没有明显的细胞毒性。其中5、10、20 ug/mL的EMP-1可显著促进细胞增殖，因此测定用于干预细胞的EMP-1浓度为5、10和20 ug/mL用于后续实验。
实施例12：选择800 mmol/L乙醇损伤细胞12 h建立酒精诱导的急性胃黏膜损伤模型。然后分别加入新疆大果沙枣EMP-1浓度为0 ug/mL、2.5 ug/mL、5 ug/mL、10 ug/mL、20ug/mL和25 ug/mL孵育3 h，吸出废液，每孔加入10 μL CCK8溶液。继续孵育1h后，在450 nm处测定吸光度。计算不同浓度酒精刺激下的细胞增殖率。然后，在安全剂量范围内，基于模拟酒精刺激计算EMP-1干预后的细胞增殖率。
实施例13：根据分组结果处理GES-1细胞。吸出废液，用DCFH-DA(2,7-二氯-氢荧光素双乙酸酯)(在37 °C下无光照孵育细胞(最终浓度为10 µM，溶液为无FBS的RPMI 1640培养基) 30分钟，然后用无FBS的RPMI 1640培养基洗涤两次，在荧光显微镜下观察活性氧ROS的分布情况。接着用同样的处理方法，使用流式细胞术检测细胞内活性氧(ROS)水平。如图13所示正如预期的那样，随着EMP-1的干预细胞形态逐渐由分散的椭圆形恢复成规则的铺路梭形，细胞产生的荧光逐渐减少。通过流式细胞术表明乙醇暴露组ROS积累显著高于对照组，其ROS水平是对照组的2.11倍。EMP-1以浓度依赖性方式显着降低了乙醇介导的ROS活化。
实施例14-16：根据分组结果处理GES-1细胞。吸出废液，使用500 μL PBS再次悬浮细胞。细胞被超声波以300W的功率打破。使用BCA试剂盒检测细胞的蛋白质浓度，再使用超氧化物歧化酶SOD试剂盒，过氧化氢酶CAT试剂盒和谷胱甘肽过氧化物酶GSH-Px试剂盒分别检测每组的SOD、CAT和GSH-Px含量。如图14所示与对照组（p<0.5）相比，乙醇暴露组中SOD、CAT和GSH-px的活性显着降低，EMP-1预处理显著（P<0.05）使SOD、CAT和GSH-px活性升高且呈剂量反应关系，表明EMP-1具有抗氧化活性。
实施例17-18：丙二醛MDA不仅具有细胞毒性，会引起蛋白质、核酸等生命大分子的交联聚合，还会使细胞膜结构和功能上受到不可逆损伤。髓过氧化物酶（MPO）是一种储存在中性粒细胞中的酶，是胃肠道损伤和中性粒细胞浸润的标志物。MPO在炎症过程中会释放到细胞外液中，当MPO缺陷的中性粒细胞因过量的注入炎症部位而级联发生氧化应激。因此，根据分组结果处理GES-1细胞。吸出废液，使用试剂盒内的裂解液再次悬浮细胞。细胞被超声波以300W的功率打破。使用BCA试剂盒检测细胞的蛋白质浓度，而分别使用丙二醛MDA试剂盒和髓过氧化物酶MPO试剂盒检测每组的MDA和MPO含量。乙醇诱导后MDA和MPO显著增加（p<0.05）。EMP-1处理后显著（P<0.05）使MDA和MPO活性降低且呈剂量反应关系，上述指标的变化表明，EMP-1可以通过剂量依赖性减少MDA和MPO来提高抗氧化能力。
Claims (5)
1.一种新疆大果沙枣均一性多糖EMP-1，其特征在于：EMP-1的相对分子量为13.80kDa，主要由不同比例的鼠李糖、阿拉伯糖、半乳糖、葡萄糖、木糖和半乳糖醛酸组成，摩尔比为12.7%：16.1%：38.7%：11.8%：11.2%：9.6%。
2.一种如权利要求1所述新疆大果沙枣多糖的制备方法，其特征在于依次按照如下步骤进行：
a . 原料粉碎：将新鲜大果沙枣经除杂、清洗、于太阳下晾晒一天除去大部分水分，后置于50 ℃烘箱干燥过夜， 烘干至重量恒定且水分含量5 %以下。人工将果肉和果核分离，用粉碎机粉碎后，过100目筛将其转化成细粉；
b. 热水提取：取大果沙枣细粉与去离子水(1:30 g/mL)混合，90 ℃搅拌提取3小时，重复提取三次。抽滤后合并提取液，通过真空旋转蒸发程序将提取液浓缩，得浓缩液；
c . 醇沉：在浓缩液中加入四倍体积的95%乙醇沉淀24 h。沉淀产物用丙酮和乙醚洗涤三次，经冷冻干燥得到大果沙枣粗多糖；
d . 脱色素：采用大孔树脂D-101脱色素。先用无水乙醇将大孔树脂浸泡24 h备用，采用高60 cm，内直径3 cm规格的层析柱，装柱时采用湿法上柱，用去离子水反复洗脱大孔树脂直至滴出液体无浑浊无醇味，将沙枣粗多糖溶液缓慢倒入层析柱中进行洗脱去除色素。真空旋蒸至适量体积，经冷冻干燥后得到脱色素粗多糖；
e . 脱蛋白：采用酶法脱蛋白。简而言之，将大果沙枣粗多糖溶解于去离子水中，55 ℃与木瓜蛋白酶反应2 h，持续搅拌。100 ℃沸水浴10 min后离心去除变性蛋白。将上清液浓缩后冷冻干燥，得到脱蛋白多糖；
f . 离子交换柱层析：将脱色和脱蛋白的沙枣多糖溶于超纯水中，载于DEAE-52色谱柱(3 cm × 60 cm)上。用超纯水平衡2 h后，用0.3 mol/L的NaCl溶液梯度洗脱，流速为2 mL/min，每管10 mL，苯酚-硫酸法在线监测。根据洗脱曲线自动采集组分。真空旋蒸浓缩，透析脱盐(3500 Da)后冷冻干燥，最终得到多糖粗品；
g. 凝胶过滤柱层析：Sephadex G-100凝胶色谱柱以双蒸水洗脱多糖粗品，浓缩冷冻干燥后获得一种多糖纯品，命名为大果沙枣纯化多糖EMP-1。
3.权利要求1所述的新疆大果沙枣多糖EMP-1在制备具有免疫调节作用的药物或保健品中的用途。
4.根据权利要求3所述的用途，其特征在于：所述的药物或保健品是促进细胞增殖的药物或保健品。
5.根据权利要求3所述的用途，其特征在于：所述的药物或保健品是缓解细胞氧化应激的药物或保健品。
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