CN115746156A - 一种具有免疫调节功能的枸杞多糖及其制备方法 - Google Patents
一种具有免疫调节功能的枸杞多糖及其制备方法 Download PDFInfo
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- CN115746156A CN115746156A CN202211477239.9A CN202211477239A CN115746156A CN 115746156 A CN115746156 A CN 115746156A CN 202211477239 A CN202211477239 A CN 202211477239A CN 115746156 A CN115746156 A CN 115746156A
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- lycium barbarum
- ethanol
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Abstract
本发明公开了一种具有免疫调节功能的枸杞多糖及其制备方法。该多糖采用80%乙醇提取去除小分子物质、Sevage法脱除蛋白以及大孔树脂脱色,经DEAE离子交换和SephacrylS‑100凝胶渗透色谱分离纯化得到枸杞多糖。该多糖由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成。通过部分酸水解和寡糖测序分析确定了枸杞多糖的骨架结构,核磁共振谱图归属其侧链结构信息。本发明制备得到的枸杞多糖可增强RAW264.7细胞增殖活性,上调细胞因子和炎症介质的释放以及mRNA的表达,激活MAPK通路发挥免疫调节作用。该枸杞多糖可作为免疫佐剂及免疫增强功能性食品或药品。
Description
技术领域
本发明属于生物医药领域,主要涉及一种具有免疫调节作用的多糖及其制备方法。
背景技术
免疫系统是机体抵御外来病原体的重要系统,当病原微生物入侵宿主时,参与机体非特异性免疫的巨噬细胞合成并分泌趋化因子和细胞因子,增强机体抵抗力。传统中草药多糖以其多种途径、多靶点对免疫细胞具有良好的激活作用而受到广泛关注。传统中草药多糖具有可以增强巨噬细胞吞噬能力,诱导炎症因子(如肿瘤坏死因子-α、白细胞介素-1β、和白细胞介素-6)和炎症介质(如一氧化氮)表达间接杀死病原体,从而参与免疫调节信号通路。
枸杞子是一种具有数千年使用历史的补益中药,色彩鲜艳悦目,品之香甜可口。作为一种倍受青睐的餐桌美食和滋补佳品在世界范围内广泛使用,以各种形式出现在果汁、酒、茶和各种固体食品中。多糖类资源性物质是枸杞子中最著名的生物活性成分,具有多种药用特性。与此同时,免疫调节作用是最主要的,并参与其他多种活性。然而枸杞多糖的化学结构和组成多样,细微的结构变化可能影响其免疫活性。枸杞多糖生物活性受其单糖组成、分子量、糖苷键和分支程度等多方面影响。为了探索枸杞多糖结构与免疫活性之间的关系,本发明对枸杞多糖精细结构进行表征及探索其免疫活性机制,为枸杞子免疫增强应用的开发提供一定的理论基础。
发明内容
本发明提供一种枸杞多糖及其在免疫调节中的应用,该枸杞多糖是从枸杞子中制备得到的中性阿拉伯半乳聚糖,体外RAW264.7巨噬细胞活性试验表明,该多糖具有显著的细胞增殖能力,促进NO、TNF-α和IL-6等细胞因子的分泌,并激活MAPK信号通路。因此,所述枸杞多糖具有免疫调节的前景,有望用于增强免疫力的食品及保健品添加剂。
一种具有免疫调节功能的枸杞多糖(简称LFP-80-W1),所述枸杞多糖由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖构成,所述枸杞多糖的分子量为4.58×104Da。
作为优选方案,以上所述的具有免疫调节功能的枸杞多糖,所述枸杞多糖由含量为2.97%、44.99%、3.65%、1.06%、6.48%、40.85%的鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖构成。
本发明所述的具有免疫调节功能的枸杞多糖的制备方法,包括以下步骤:
(1)取枸杞子,加稀乙醇提取,得到稀乙醇提取液和醇提药渣;
(2)取步骤(1)醇提药渣加去离子水提取,将所得水提液进行浓缩,得到水溶性组分;
(3)将所述水溶性组分,先用低浓度乙醇进行沉淀后,收集溶液部分,再用高浓度乙醇进行醇沉,收集沉淀,用乙醇洗涤后,冷冻干燥,得沉淀产物;
(4)将步骤(3)沉淀产物溶于水中,先经Sevag法脱蛋白、然后采用D101大孔树脂脱色,最后使用透析袋进行透析,得透析产物,冷冻干燥;
(5)将干燥产物再次溶于水中,经离子交换树脂洗脱,收集纯水洗脱组分,减压浓缩后重新溶于纯水中,上样至聚丙烯酰胺葡聚糖凝胶柱反复分离,收集洗脱峰组分,冷冻干燥,即得。
作为优选方案,以上所述的具有免疫调节功能的枸杞多糖的制备方法,其特征在于,包括以下步骤:
(1)取枸杞子,加体积浓度70~80%的乙醇提取1~3次,得到醇提液和醇提药渣;
(2)取醇提药渣加6~20倍的去离子水提取1~2次,将所得水提液进行浓缩,得到水溶性组分;
(3)将所述水溶性组分,加入乙醇调整至醇体积浓度为30%,沉淀后,收集溶液部分,减压浓缩,加入乙醇,使其最终乙醇体积浓度达到80%,收集沉淀,用乙醇洗涤,冷冻干燥得沉淀产物;
(4)将沉淀产物溶于水中,先经Sevage法,即体积比为3:1的三氯甲烷:正丁醇溶液进行脱蛋白、然后采用D101大孔树脂脱色,最后使用3500Da透析袋进行透析,得透析产物,冷冻干燥;
(5)将步骤.(4)冷冻干燥产物再次溶于水中,经离子交换树脂洗脱,收集纯水洗脱组分,减压浓缩后重新溶于纯水中,上样至Sephacryl S-100凝胶柱反复分离,收集洗脱峰组分,冷冻干燥,即得。
作为优选方案,以上所述的具有免疫调节功能的枸杞多糖的制备方法,所述的步骤(1),取枸杞子,加体积浓度80%的乙醇回流提取或连续回流提取2~3次,每次1~2小时,得到80%的乙醇提取液和醇提药渣;
所述的步骤(2),取醇提药渣加6~15倍的水煎煮或回流提取1~2次,每次1~2小时,将所得水提液进行浓缩,得到水溶性组分。
作为优选方案,以上所述的具有免疫调节功能的枸杞多糖的制备方法,步骤(4)中的D101大孔树脂脱色,吸附时间为20min,洗脱流速为2.4BV/h,洗脱溶剂为去离子水;使用3500Da透析袋常温透析24h,每4h更换一次透析液。
作为优选方案,以上所述的具有免疫调节功能的枸杞多糖的制备方法,步骤(5)所述的离子交换树脂型号为DEAE-52,枸杞多糖吸附30min后,使用去离子水洗脱;所述的聚丙烯酰胺葡聚糖凝胶型号为Sephacryl S-100,使用去离子水反复洗脱。
本发明制备得到的枸杞多糖,扫描电镜形貌特征分析为不规则的薄片状,质地松散,原子力显微镜显示所述多糖由随机线性链和少量球形聚集组成,在三维图像中具有不同高度的锥形形状。
更进一步的,所述枸杞多糖经甲基化气相质谱检测的糖醇乙酰酯峰为11个,其中→3,6)-Galp-(1→糖苷键含量最高。
更进一步的,所述枸杞多糖的1H-NMR图谱中,位于δ4.35-5.27ppm处有11个端基氢质子,13C-NMR图谱中,δ100.68-109.17ppm范围内对应11个端基碳质子。
更进一步的,所述枸杞多糖经70℃部分酸水解的寡糖图谱由15个聚合度依次增加的寡糖组成,包括两对同分异构体。
有益效果:本发明制备得到的枸杞多糖可促进RAW264.7细胞增殖,促进NO、IL-6和TNF-α的释放以及上调相应mRNA的表达,激活MAPK信号通路促进磷酸化p38、p-ERK及p-JNK蛋白水平表达。
本发明提供的枸杞多糖可用于免疫增强产品,包括但不限于疫苗佐剂、功能性食品、免疫用途的保健品或药品。
附图说明
图1为枸杞多糖LFP-80-W1在DEAE-52纤维素柱和Sephacryl S-100凝胶柱分离纯化图。
图2中A为枸杞多糖LFP-80-W1高效凝胶分析图谱;B为枸杞多糖LFP-80-W1单糖组成图谱。
图3中A为枸杞多糖LFP-80-W1的扫描电镜图;B为LFP-80-W1的原子力显微图。
图4为枸杞多糖LFP-80-W1的甲基化图谱。
图5为枸杞多糖LFP-80-W1的核磁图谱。
图6为枸杞多糖LFP-80-W1的ABEE标记的低聚糖总离子图。
图7为枸杞多糖LFP-80-W1对RAW264.7细胞活力、NO、IL-6和TNF-α产生的影响。
图8为枸杞多糖LFP-80-W1对iNOS、IL-6和TNF-αmRNA表达水平的影响。
图9为枸杞多糖LFP-80-W1对RAW 264.7细胞MAPK信号通路磷酸化蛋白的影响。
具体实施方式
本发明采用RAW264.7巨噬细胞进行体外活性筛选评价试验,从枸杞总多糖中筛选发现了一种具有潜在免疫调节作用的枸杞多糖组分。
以下对本发明技术方案的具体实施方式详细描述。
实施例1:制备具有潜在免疫调节作用的枸杞多糖LFP-80-W1
1、枸杞多糖组分LFP-80-W1的制备方法
1.1仪器、试剂与材料
枸杞子干果、DEAE-52纤维素、Sephacryl S-100、葡聚糖标准品、单糖标准品、氰化硼氢化钠、对氨基苯甲酸乙酯。所有其他化学物质为分析级。真空冷冻干燥机、紫外分光光度计、电子分析天平、离心机、高效液相色谱与示差折光检测器(RID)系统。
1.2LFP-80-W1的提取分离流程
取10kg干燥枸杞子用80%乙醇回流提取2次,每次2小时,提取后的枸杞残渣加20倍水在100℃回流提取2次,每次2小时,合并提取液,减压浓缩,浓缩液加入无水乙醇调整至醇体积浓度为30%,以沉淀纤维、果胶等不溶性成分后得到LFP-30溶液组分。随后,浓缩LFP-30溶液组分,加入乙醇,使其最终乙醇体积浓度达到80%,4℃沉淀过夜,获得LFP-80组分,冷冻干燥。
将LFP-80组分再次溶于水中,加入Sevage试剂(体积比为3:1的三氯甲烷:正丁醇溶液)脱蛋白,经D101大孔树脂吸附20min后,用去离子水洗脱,收集洗脱液,得到除色素LFP-80组分。取2g纯化后的LFP-80组分重溶于水,在DEAE-52阴离子交换柱(4.5cm×60cm)上分离,吸附30min后,使用去离子水洗脱,苯酚硫酸法检测收集洗脱组分。用Sephacryl S-100凝胶渗透色谱柱(150cm×2.6cm)进行纯化,去离子水洗脱,洗脱液采用苯酚-硫酸法进行监测,收集目标组分,浓缩后重复上述凝胶渗透色谱分离操作,得到目标多糖LFP-80-W1。
图1是枸杞多糖组分LFP-80-W1在DEAE-52纤维素和Sephacryl S-100凝胶洗脱曲线。2、枸杞多糖LFP-80-W1组成分析
2.1、枸杞多糖LFP-80-W1糖醛酸、总多糖和蛋白含量的测定分析:分别配制2mg/mL枸杞多糖LFP-30、LFP-80和LFP-80-W1组分,采用苯酚硫酸法测定总糖含量,以葡萄糖为标准品,绘制标准曲线为y=15.898x+0.0037,R2=0.999;采用间羟基联苯法测定糖醛酸含量,以半乳糖醛酸为标准品,绘制标准曲线为y=8.3825x+0.0266,R2=0.999;采用BCA蛋白测定试剂盒测定蛋白质含量。上述LFP-30、LFP-80和LFP-80-W1组分的理化性质测定结果如表1。
表1:LFP-30、LFP-80和LFP-80-W1组分糖醛酸、总多糖和蛋白含量表
LFP-30 | LFP-80 | LFP-80-W1 | |
得率(%) | 1.32 | 1.67 | 0.125 |
糖醛酸(%) | 26.35 | 35.44 | - |
总多糖(%) | 44.71 | 20.08 | 94.5 |
蛋白(%) | 27.63 | 22.17 | 1.7 |
2.2、枸杞多糖LFP-80-W1纯度和分子量测定:配制2mg/mL以上制备得到的枸杞多糖LFP-80-W1 PBS的缓冲盐溶液,采用高效凝胶渗透色谱法(HPGPC)配备示差折光检测器(RID)测定分子量,用0.01M PBS缓冲液以0.4mL/min的流速洗脱样品。此外,以葡聚糖为标品绘制的校准曲线y=-6.4299x+67.77,R2=0.997,估算重均分子量。如图2所示,LFP-80-W1在HPGPC上表现为连续对称的单峰,计算其分子量为4.58×104Da。
2.3、枸杞多糖LFP-80-W1单糖组成测定:取5mg LFP-80-W1用2M TFA在110℃下水解2h,将释放的单糖混合物与30mg硼氢化钠混合,再加入0.5mL吡啶和0.5mL醋酸酐将单糖转化为相应的糖醇乙酰酯,进入气相质谱分析,程序升温条件为:初始温度100℃,保持3min,以20℃/min升至200℃后,保持2min,以5℃/min升至230℃后,保持2min,以10℃/min升至280℃后,保持8min。结果表明,LFP-80-W1由鼠李糖2.97%、阿拉伯糖44.99%、木糖3.65%、甘露糖1.06%、葡萄糖6.48%和半乳糖40.85%组成,其中阿拉伯糖和半乳糖为主要单糖。
2.4、枸杞多糖LFP-80-W1形貌特征分析:通过扫描电镜和原子力显微镜是分析多糖的形态特征。扫描电镜形貌特征分析LFP-80-W1为不规则的薄片状,质地松散,说明LFP-80-W1存在复杂的多糖分支结构。原子力显微镜显示LFP-80-W1由随机线性链和少量球形聚集组成,在三维图像中具有不同高度的锥形形状,表明分子内和分子间的范德华力和氢键作用导致LFP-80-W1分子自组装成聚集结构。
2.5、枸杞多糖LFP-80-W1甲基化分析:将5mg真空干燥的LFP-80-W1样品溶解在4mLDMSO中,用500mgNaOH和0.5mL碘甲烷处理。用氯仿萃取甲基化多糖,将完全甲基化产物转化为相应的部分甲基化糖醇乙酸酯(PMAAs)衍生物。PMAAs在110℃下用2M TFA水解2小时,NaBH4还原,用吡啶和乙酸酐乙酰化。通过相对保留时间和GC-MS碎片模式与CCRC标准谱数据库进行比较,对乙酰化PMAAs进行分析和鉴定。如表2所示,LFP-80-W1含有11个部分甲基化的乙酸乙二醇酯峰。高含量→3,6)-Galp-(1→残基表明LFP-80-W1可能是一种高度支化的多糖。
表2LFP-80-W1的糖苷键组成
枸杞多糖LFP-80-W1核磁共振分析:约30mg LFP-80-W1溶解在氧化氘中进行核磁共振分析。枸杞多糖LFP-80-W1的1H-NMR图谱中,如图5所示,位于δ4.35-5.27ppm处有11个端基氢质子,13C-NMR图谱中,δ100.68-109.17ppm范围内对应11个端基碳质子,说明α构型和β构型均存在。化学位移δ4.96,4.99,5.13和5.27来自于α-L-阿拉伯糖,化学位移δ4.61/100.68归属于末端β-D-葡萄糖和半乳糖,δ4.35和4.38处的信号来源于1,3-β-D-半乳糖、1,6-β-D-半乳糖和1,3,6-β-D-半乳糖,δ4.99为1,4-α-D-半乳糖。
枸杞多糖LFP-80-W1寡糖序列分析:多糖水解成可测量的寡糖是表征多糖结构的关键组成部分。将4mg LFP-80-W1用2mL的1mol/L TFA在70℃下部分水解2小时,水解样品与衍生化试剂(0.6mol/LABEE、冰醋酸和1.4mol/LABEE)混合,将ABEE标记的低聚糖产物溶于50%甲醇中,采用ACQUITYUHPLC系统结合SYNAPT TM Q-TOF检测器进行分析。
如图6所示,在色谱图中显示出15个分离良好的峰。精确质量(m/z)表明,它们代表了一个聚合度依次增加的寡糖序列。峰1代表末端阿拉伯糖,峰2和3为半乳二糖同分异构体,峰4和5为半乳三糖同分异构体。其余峰的寡糖谱为4-11DP的线性1,6-β-D-半乳糖,每个半乳糖单元是由C-3位损失CH2O(30Da)片段形成的。LFP-80-W1的寡糖裂解信息总结为表3。
表3 LFP-80-W1的UHPLC-Q-TOF/MS质谱信息
编号 | 保留时间(min) | [M+H]<sup>+</sup> | 分子式 | 聚合度 |
1 | 9.079 | 300.1455 | C<sub>14</sub>H<sub>22</sub>NO<sub>6</sub> | 1 |
2 | 8.521 | 432.1874 | C<sub>19</sub>H<sub>30</sub>NO<sub>10</sub> | 2 |
3 | 8.324 | 432.1876 | C<sub>19</sub>H<sub>30</sub>NO<sub>10</sub> | 2 |
4 | 7.828 | 564.2288 | C<sub>24</sub>H<sub>38</sub>NO<sub>14</sub> | 3 |
5 | 7.747 | 564.2297 | C<sub>24</sub>H<sub>38</sub>NO<sub>14</sub> | 3 |
6 | 7.487 | 696.2696 | C<sub>29</sub>H<sub>46</sub>NO<sub>18</sub> | 4 |
7 | 7.136 | 828.3138 | C<sub>34</sub>H<sub>54</sub>NO<sub>22</sub> | 5 |
8 | 6.816 | 960.3547 | C<sub>39</sub>H<sub>62</sub>NO<sub>26</sub> | 6 |
9 | 6.611 | 1092.3964 | C<sub>44</sub>H<sub>70</sub>NO<sub>30</sub> | 7 |
10 | 6.425 | 1224.4402 | C<sub>49</sub>H<sub>78</sub>NO<sub>34</sub> | 8 |
11 | 6.281 | 1356.4861 | C<sub>54</sub>H<sub>86</sub>NO<sub>38</sub> | 9 |
12 | 5.835 | 1488.5190 | C<sub>59</sub>H<sub>94</sub>NO<sub>42</sub> | 10 |
13 | 5.701 | 1620.5608 | C<sub>64</sub>H<sub>102</sub>NO<sub>46</sub> | 11 |
14 | 5.174 | 1752.5957 | C<sub>69</sub>H<sub>110</sub>NO<sub>50</sub> | 12 |
15 | 5.061 | 1884.6046 | C<sub>74</sub>H<sub>118</sub>NO<sub>54</sub> | 13 |
。
实例2枸杞多糖的免疫调节活性
1、LFP-80-W1对RAW264.7细胞增殖的影响
将RAW 264.7细胞以1×105cells/mL的密度接种于96孔板中,孵育24小时。然后将细胞添加于一系列浓度的LFP-80-W1(0、25、50、100、200和400mg/mL)的无血清RPMI-1640中24小时。用CCK-8法检测细胞活力,采用酶标仪在450nm处测量各孔的吸光度。以相同体积的细胞培养液作为空白对照。
图7为LFP-80-W1对RAW264.7细胞增殖的影响。LFP-80-W1给药24h后,25~400μg/mL浓度范围内RAW 264.7细胞存活率显著高于对照组,且呈剂量无关关系(P<0.05)。当LFP-80-W1浓度达到100μg/mL时,对细胞活力的影响最为显著,当浓度开始超过100μg/mL时,细胞活力开始下降。
2、枸杞多糖LFP-80-W1对RAW264.7细胞的NO、IL-6和TNF-α分泌量的测定
将对数生长期的RAW 264.7细胞以2.0×105cells/mL的浓度接种到12孔板上,然后用脂多糖(LPS;1μg/mL)或LFP-80-W1以不同浓度(10、30、100μg/mL)培养24h或48h。培养上清液中的NO水平用等体积的Griess试剂混合。根据制造商的说明,收集细胞培养上清,加入ELISA试剂测定了TNF-α和IL-6的值。
图7纵坐标代表RAW264.7细胞的NO、TNF-α和IL-6的分泌量,实验结果表明LFP-80-W1(10、30和100μg/mL)无论是处理24h还是48h,都能以剂量依赖性的方式促进NO的产生(图7B)。如图7C所示,与对照组相比,不同浓度的LFP-80-W1和LPS产生的TNF-α含量显著升高(P<0.05)。30和100μg/mL LFP-80-W1和LPS在不同时间处理细胞时IL-6增强(图7D)。
3、Real-Time PCR检测iNOS、IL-6和TNF-α的mRNA表达水平。
细胞以5×105Cells/mL的浓度种于6孔板中。在无酶环境中,使用RNeasy MiniKit提取总RNA。使用PrimeScriptTMRT Master Mix将约1μg RNA反转录为cDNA。以GAPDH基因mRNA作为对照确定Ct值。所有反应重复3次,用2-ΔΔCT方法计算相对基因表达数。所用引物见表4。
表4引物序列
基因 | 上游引物序列 | 下游引物序列 |
GAPDH | GGTTGTCTCCTGCGACTTCA | TGGTCCAGGGTTTCTTACTCC |
iNOS | ATCTTGGAGCGAGTTGTGGATTGTC | TAGGTGAGGGCTTGGCTGAGTG |
IL-6 | CTCCCAACAGACCTGTCTATAC | CCATTGCACAACTCTTTTCTCA |
TNF-α | ATGTCTCAGCCTCTTCTCATTC | GCTTGTCACTCGAATTTTGAGA |
如图8所示,在与LFP-80-W1或LPS孵育24h后,iNOS、TNF-α和IL-6的mRNA表达水平显著增强,与ELISA检测结果一致。LFP-80-W1对IL-6细胞因子释放的刺激最为明显。同时,实验发现LFP-80-W1诱导的iNOS等细胞因子mRNA表达低于LPS模型对照组。因此,这些结果表明,LFP-80-W1刺激NO和炎症因子的释放在特定的安全范围内,并没有达到引起炎症的效果。
4、LFP-80-W1对RAW264.7细胞MAPK通路的影响
以5×105Cells/mL的浓度种于6孔板中。蛋白质样本用含有蛋白酶和磷酸酶抑制剂的RIPA缓冲液进行裂解。用含5%脱脂牛奶的TBST阻断细胞膜1小时,加入ERK1/2、p-ERK1/2、p38、p-p38、JNK、p-JNK的一抗(1:1000),再用二抗(1:5000)育1小时。采用Bio-Rad化学发光成像系统检测抗体信号。利用ImageJ软件对条带强度进行量化和归一化。
MAPK信号通路是细胞增殖、分化、细胞凋亡的关键信号通路,磷酸化激活MAPK后,进入细胞核内调节转录调控。MAPK的ERK、JNK和p38激酶可能在炎症和凋亡中发挥重要作用。如图9所示,当10、30和100μg/mL的LFP-80-W1或LPS处理细胞时,MAPK通路p-p38、p-ERK和p-JNK的蛋白活化水平均被促进。此外,LFP-80-W1在100μg/mL时引起的ERK、JNK和p38磷酸化水平最高,与LPS组相似。因此,这些结果表明,LFP-80-W1可以激活RAW 264.7细胞MAPK信号通路关键蛋白的磷酸化水平。
本发明方案所公开的技术手段不仅限于上述技术手段所公开的技术手段,还包括由以上技术特征等同替换所组成的技术方案。本发明的未尽事宜,属于本领域技术人员的公知常识。
Claims (8)
1.一种具有免疫调节功能的枸杞多糖,其特征在于,所述枸杞多糖由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖构成,所述枸杞多糖的分子量为4.58×104Da。
2.根据权利要求1所述的具有免疫调节功能的枸杞多糖,其特征在于,所述枸杞多糖由质量百分含量为2.97%、44.99%、3.65%、1.06%、6.48%、40.85%的鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖构成。
3.权利要求1或2所述的具有免疫调节功能的枸杞多糖的制备方法,其特征在于,包括以下步骤:
(1)取枸杞子,加稀乙醇提取,得到稀乙醇提取液和醇提药渣;
(2)取步骤(1)醇提药渣加水提取,将所得水提液进行浓缩,得到水溶性组分;
(3)将所述水溶性组分,先用低浓度乙醇进行沉淀后,收集溶液部分,再用高浓度乙醇进行醇沉,收集沉淀,用乙醇洗涤后,冷冻干燥,得沉淀产物;
(4)将步骤(3)沉淀产物溶于水中,先经Sevage法脱蛋白、然后采用D101大孔树脂脱色,最后使用透析袋进行透析,得透析产物,冷冻干燥;
(5)将干燥产物再次溶于水中,经离子交换树脂洗脱,收集纯水洗脱组分,减压浓缩后重新溶于纯水中,上样至聚丙烯酰胺葡聚糖凝胶柱反复分离,收集洗脱峰组分,冷冻干燥,即得。
4.根据权利要求3所述的具有免疫调节功能的枸杞多糖的制备方法,其特征在于,包括以下步骤:
(1)取枸杞子,加体积浓度70~80%的乙醇提取1~3次,得到醇提液和醇提药渣;
(2)取醇提药渣加6~20倍的去离子水提取1~2次,将所得水提液进行浓缩,得到水溶性组分;
(3)将所述水溶性组分,加入乙醇至醇体积浓度至30%,沉淀后,收集溶液部分,随后减压浓缩,加入乙醇,使其最终乙醇体积浓度达到80%,收集沉淀,乙醇洗涤,冷冻干燥得沉淀产物;
(4)将沉淀产物溶于水中,先经Sevage法,即体积比为3:1的三氯甲烷:正丁醇溶液进行脱蛋白、然后采用D101大孔树脂脱色,最后使用3500Da透析袋进行透析,得透析产物,冷冻干燥;
(5)将步骤.(4)冷冻干燥产物再次溶于去离子水中,经离子交换树脂洗脱,收集纯水洗脱组分,减压浓缩后重新溶于去离子水中,上样至聚丙烯酰胺葡聚糖凝胶柱反复分离,收集洗脱峰组分,冷冻干燥,即得。
5.根据权利要求4所述的具有免疫调节功能的枸杞多糖的制备方法,其特征在于,所述的步骤(1),取枸杞子,加体积浓度80%的乙醇回流提取或连续回流提取2~3次,每次1~2小时,得到80%的乙醇提取液和醇提药渣;
所述的步骤(2),取醇提药渣加6~15倍的水煎煮或回流提取1~2次,每次1~2小时,将所得水提液进行浓缩,得到水溶性组分。
6.根据权利要求3或4所述的具有免疫调节功能的枸杞多糖的制备方法,其特征在于,步骤(4)中的D101大孔树脂脱色,吸附时间为20min,洗脱流速为2.4BV/h,洗脱溶剂为去离子水;使用3500Da透析袋常温透析24h,每4h更换一次透析液。
7.根据权利要求3或4所述的具有免疫调节功能的枸杞多糖的制备方法,其特征在于,步骤(5)所述的离子交换树脂型号为DEAE-52,使用去离子水洗脱;
所述的聚丙烯酰胺葡聚糖凝胶型号为Sephacryl S-100,使用去离子水反复洗脱。
8.权利要求1或2所述的具有免疫调节功能的枸杞多糖在制备提高免疫力的保健品或药品中的应用。
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