CN115124629B - 一种海藻多糖钙的制备及其应用 - Google Patents
一种海藻多糖钙的制备及其应用 Download PDFInfo
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Abstract
本发明属于海藻多糖研究领域，公开了一种具有抑制α‑葡萄糖苷酶活性的裙带梗多糖UPPS‑1及其制备方法和应用。UPPS‑1是裙带梗用氯化钙溶液浸提，经离子交换柱和凝胶柱分离纯化得到的均一多糖，重均分子量为174.88kDa。经α‑葡萄糖苷酶活性研究，UPPS‑1抑制肠黏膜上的α‑葡萄糖苷酶，使淀粉分解为葡萄糖的速度减慢，从而减少和延缓小肠对葡萄糖的吸收，改善糖尿病人血糖异常。本发明的多糖可作为潜在的糖尿病抑制剂，用于功能性食品或药物的开发。
Description
技术领域
本发明涉及一种海藻提取物的制备方法及其应用。
背景技术
褐藻含有多糖、蛋白质、维生素、矿物质等多种营养成分。多糖是一类具有抗氧化、抗肿瘤、免疫调节、抗病毒等多种生物活性的高分子化合物，因此在药物开发上具有应用价值。
裙带梗是裙带菜的梗段部分，是一种大型经济褐藻,在我国主要分布在辽宁、山东、江苏、浙江等地，属于褐藻门，褐子纲。含水量大，组织较硬，色泽浓褐，因其口感佳广泛用于食品烹饪。裙带菜在抗病毒、抗肿瘤、降血压、免疫调节以及心脑血管疾病治疗等方面具有很好的功效。目前对裙带梗多糖的提取分离及生物活性未做深入研究。
发明内容
本发明的首要目的在于提供一种具有调节糖尿病的裙带梗多糖。
本发明的另一目的在于提供上述具有调节糖尿病裙带梗多糖的制备方法；本发明以裙带梗实体为研究对象，通过氯化钙浸提和醇沉，经离子交换柱以及分子筛进行分离纯化并研究其生物活性，解析了裙带梗多糖的分子量及单糖组成。
本发明的再一目的在于提供上述具有调节糖尿病的裙带梗多糖的应用。
本发明的目的通过下述技术方案实现：
一种具有调节糖尿病的裙带梗多糖UPPS-1，重均分子量为174.880kDa
所述点裙带梗多糖的糖含量为37.95％。
所述点裙带梗多糖UPPS-1主要由摩尔比为4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01的甘露糖醛酸、氨基葡萄糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、岩藻糖组成。
上述的点裙带梗多糖的制备方法，包括以下步骤：
(1)裙带梗脱盐，晒干打成粉末。
(2)加入CaCl2溶液进行提取，收集上清液，减压浓缩。
(3)将浓缩液经乙醇醇沉、脱色、除蛋白、透析、冷冻干燥得到粗多糖。
(4)取粗多糖配置成溶液经离子交换柱洗脱，苯酚硫酸法跟踪监测搜集目标峰产物，经浓缩、透析、冷冻干燥后获得粗分组分。
(5)将粗分组分经凝胶柱进一步分离，苯酚硫酸法跟踪监测搜集目标峰产物，经浓缩、透析、冷冻干燥后获得细分组分。
所述步骤(1)具体按照以下步骤：将裙带梗用自来水清洗表面的盐，每12h更换一次水，浸泡三天后沥干，自然晒干后粉粹，得到裙带梗粉末。
所述步骤(2)具体按照以下步骤：将裙带梗粉末按0.15mol/LCaCl2，料液比1：15，70℃下提取3h。收集滤液，减压浓缩，得到裙带梗浓缩液。
所述步骤(3)具体按照以下步骤：将裙带梗浓缩液使用80％乙醇醇沉过夜，离心收集沉淀后用少量去离子水复溶，氯化717阴离子树脂55℃脱色12h，Sevage法除蛋白(氯仿：正丁醇＝4:1)三次以上，直至两相分界处无白色絮状物。3500Da透析48h、冷冻干燥得到粗多糖。
所述步骤(4)具体按照以下步骤：将裙带梗粗多糖用去离子水溶解，配置成浓度为30mg/mL的多糖溶液经DEAE-52离子交换柱分离，依次使用0、0.5、1、2mol/LNacl溶液洗脱，流速控制在1mL/min，洗脱时间10min/管，采用苯酚硫酸法跟踪监测，根据吸光度收集主峰部位。收集0.5mol/LNacl溶液洗脱组分，浓缩后用3500Da透析袋透析48h、冷冻干燥得到粗分裙带梗多糖UPPS-1。
所述步骤(5)具体按照以下步骤：将裙带梗粗分多糖用纯水溶解，配置成浓度为30mg/mL的多糖溶液经Sephadex-G100凝胶柱分离，使用纯水洗脱，流速控制在0.5mL/min，洗脱时间10min/管，采用苯酚硫酸法跟踪监测，根据吸光度收集主峰部位。收集纯水洗脱组分，减压浓缩后用3500Da透析袋透析48h、冷冻干燥得到细分裙带梗多糖UPPS-1。
上述的点裙带梗多糖UPPS-1在制备调节糖尿病中的应用。
所述α-葡萄糖苷酶抑制剂，通过抑制肠黏膜上的α-葡萄糖苷酶，使淀粉分解为葡萄糖的速度减慢，从而减少和延缓小肠对葡萄糖的吸收，改善糖尿病人血糖异常。
上述方法中，与传统的水溶剂提取方法相比，本发明的裙带梗多糖通过氯化钙提取减少了褐藻胶的生成，提高了溶解性。
本发明相对于现有技术具有如下的优点及有益效果：
1、本发明以裙带梗为研究对象，通过氯化钙溶液浸提，经过DEAE-52离子交换柱和分子筛进行分离纯化制备出裙带梗多糖UPPS-1，并确定其具体的活性用途。
2、与传统水溶剂提取方法相比，本发明的裙带梗多糖的提取方法优势在于使用氯化钙溶液，除去了部分褐藻胶，提高了多糖的溶解性。
3、经测定，裙带梗多糖UPPS-1的重均分子量为174.880kDa，主要由甘露糖醛酸、氨基葡萄糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、岩藻糖组成。
4、裙带梗多糖UPPS、UPPS-1在100-1000μg/mL浓度范围内，可抑制α-葡萄糖苷酶，减慢淀粉分解为葡萄糖的速度，从而抑制糖尿病。
附图说明
图1是裙带梗多糖经DEAE-52离子交换柱层析洗脱图；
图2是裙带梗多糖经Sephadex-G100凝胶柱柱层析洗脱图；
图3是裙带梗多糖UPPS-1的紫外光谱图；
图4是裙带梗多糖UPPS-1的红外光谱图；
图5是标准糖出峰时间表；
图6是标准糖HPLC图；
图7是裙带梗多糖UPPS-1的单糖组成图；
图8是裙带梗多糖UPPS、UPPS-1抑制α-葡萄糖苷酶影响图；
具体实施方式
下面结合说明书附图和具体施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。在未作特别说明的情况下，本发明所采用的试剂、设备和方法均为本技术领域常规市购的试剂、设备和常规的使用方法。
实施例1
将裙带梗用自来水清洗表面的盐，每12h更换一次水，浸泡三天后沥干，自然晒干后粉粹，得到裙带梗粉末；
将裙带梗粉末按0.15mol/LCaCl2，料液比1：15，70℃下提取3h。收集滤液，减压浓缩，得到裙带梗浓缩液；
将裙带梗浓缩液使用80％乙醇醇沉过夜，离心收集沉淀后用少量去离子水复溶，氯化717阴离子树脂55℃脱色12h，Sevage法除蛋白(氯仿：正丁醇＝4:1)三次以上，直至两相分界处无白色絮状物。3500Da透析48h、冷冻干燥得到粗多糖；
将裙带梗粗多糖用去离子水溶解，配置成浓度为30mg/mL的多糖溶液经DEAE-52离子交换柱分离，依次使用0、0.5、1、2mol/LNacl溶液洗脱，流速控制在1mL/min，洗脱时间10min/管，采用苯酚硫酸法跟踪监测，根据吸光度收集主峰部位。收集0.5mol/LNacl溶液洗脱组分，浓缩后用3500Da透析袋透析48h、冷冻干燥得到粗分裙带梗多糖UPPS-1；
将裙带梗粗分多糖用纯水溶解，配置成浓度为30mg/mL的多糖溶液经Sephadex-G100凝胶柱分离，使用纯水洗脱，流速控制在0.5mL/min，洗脱时间10min/管，采用苯酚硫酸法跟踪监测，根据吸光度收集主峰部位。收集纯水洗脱组分，减压浓缩后用3500Da透析袋透析48h、冷冻干燥得到细分裙带梗多糖UPPS-1；
裙带梗多糖UPPS-1糖含量的测定
称取10mg葡萄糖，定容至100mL，移取0.1、0.2、0.4、0.6、0.8、1mL葡萄糖溶液，加蒸馏水定容至1mL。60℃水浴锅溶解苯酚，取5mL定容至100mL。加入1mL 5％苯酚溶液，摇匀。加入5mL浓硫酸，摇匀，室温反应，静置30min。于490nm处测定吸光值。以葡萄糖浓度为横坐标，吸光值为纵坐标，绘制标准曲线。将裙带梗多糖UPPS-1配置成浓度为1mg/mL的溶液，取1mL按苯酚硫酸法检测。根据标准曲线y＝6.6798x-0.034(R2＝0.9997)，计算得UPPS-1糖含量为37.95％。
裙带梗UPPS-1的紫外光谱分析
取一定量上述所得的裙带梗多糖溶于蒸馏水中，浓度为1mg/mL，在分光光度计上扫描，波长范围为200-800nm，结果如图3所示，其在260nm和280nm无特征吸收峰，表明裙带梗多糖不含核酸和蛋白质。
裙带梗UPPS-1的红外光谱分析
将2mg上述所得裙带梗多糖与50mg干燥的溴化钾粉末混匀，充分研磨后压片，在4000-400cm-1的范围内进行红外光谱扫描，结果如图4所示，在3444.20cm-1出现糖类特征吸收峰，是由O-H伸缩振动产生。2930.90cm-1处有吸收峰，是C-H伸缩振动产生。1420.07cm-1处的吸收峰是C-O的伸缩振动产生。1643.99cm-1处是C＝O的非对称伸缩振动产生的吸收峰，推断出UPPS有羧基，含有糖醛酸。1243.76cm-1处有S＝O的不对称伸缩振动，821.28cm-1处是C-O-S的不对称伸缩振动产生，UPPS可能含有硫酸基。1055.07cm-1处是C-C的伸缩振动。
裙带梗UPPS-1的单糖组成分析
取10mg上诉所得裙带梗UPPS-1，用1mol/L三氟乙酸在110℃水解6h后用0.5mol/LPMP甲醇溶液衍生化，衍生物在Agilent 1260高效液相色谱仪上进行分析。
色谱条件：BDS HYPERSIL C18色谱柱(5μm×4.6mm×250mm)，检测波长250nm，柱温30℃，流速0.8mL/min，UV检测器，流动相为磷酸盐缓冲液：乙腈(87:17)。
其中标准品为：葡萄糖、半乳糖、岩藻糖、葡萄糖醛酸、氨基葡萄糖、氨基半乳糖、甘露糖、鼠李糖、木糖、半乳糖醛酸、***糖。结果如图5、6所示。
由色谱检测结果可知，裙带梗多糖主要由摩尔比为4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01的甘露糖醛酸、氨基葡萄糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、岩藻糖组成。
裙带梗UPPS、UPPS-1的α-葡萄糖苷酶活性测定
0.2U/mLα-葡萄糖苷酶0.3mL和0.4mL不同浓度多糖样品37℃恒温水浴保温10min，加入5mmol/LPNPG溶液0.3mL继续37℃恒温水浴保温20min，在波长405nm处，测定吸光值为Ai，0.2mol/L(ph＝6.8)磷酸盐缓冲液代替样品测定吸光度为A0，磷酸盐缓冲液代替酶为Aj。α-葡萄糖苷酶抑制率的公式为：
T＝[1-(Ai-Aj)/A0]×100％，结果如图6所示，裙带梗多糖UPPS、UPPS-1在100-1000ug/mL呈现良好的抑制率。
Claims (6)
1.一种具有α-葡萄糖苷酶活性的裙带梗多糖UPPS-1，其特征在于：重均分子量为174.88kDa；所述的裙带梗多糖UPPS-1由摩尔比为4.35:1.86:2.32:4.50:0.94:2.07:48.42:16.01的甘露糖醛酸、氨基葡萄糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、岩藻糖组成。
2.根据权利要求1所述的一种具有α-葡萄糖苷酶活性的裙带梗多糖UPPS-1，其特征在于：所述裙带梗多糖UPPS-1的糖含量为37.95％。
3.根据权利要求1-2任一项所述的裙带梗多糖UPPS-1的制备方法，其特征在于，包括以下步骤：
(1)裙带梗脱盐，晒干打成粉末；
(2)加入CaCl2溶液进行提取，收集上清液，减压浓缩；
(3)将浓缩液经乙醇醇沉、脱色、除蛋白、透析、冷冻干燥得到粗多糖；
(4)取粗多糖配置成溶液经离子交换柱洗脱，苯酚硫酸法跟踪监测搜集目标峰产物，经浓缩、透析、冷冻干燥后获得粗分组分；
(5)将粗分组分经凝胶柱进一步分离，苯酚硫酸法跟踪监测搜集目标峰产物，经浓缩、透析、冷冻干燥后获得细分组分；
所述步骤(4)具体按照以下步骤：将裙带梗粗多糖用去离子水溶解，配置成浓度为30mg/mL的多糖溶液经DEAE-52离子交换柱分离，依次使用0、0.5、1、2mol/LNacl溶液洗脱，流速控制在1mL/min，洗脱时间10min/管，采用苯酚硫酸法跟踪监测，根据吸光度收集主峰部位，收集0.5mol/LNacl溶液洗脱组分，浓缩后用3500Da透析袋透析48h、冷冻干燥得到粗分裙带梗多糖UPPS-1；
所述步骤(5)具体按照以下步骤：将裙带梗粗分多糖用纯水溶解，配置成浓度为30mg/mL的多糖溶液经Sephadex-G100凝胶柱分离，使用纯水洗脱，流速控制在0.5mL/min，洗脱时间10min/管，采用苯酚硫酸法跟踪监测，根据吸光度收集主峰部位，分别收集纯水洗脱组分，减压浓缩后用3500Da透析袋透析48h、冷冻干燥得到细分裙带梗多糖UPPS-1。
4.根据权利要求3所述的裙带梗多糖UPPS-1的制备方法，其特征在于：
所述步骤(1)具体按照以下步骤：将裙带梗用自来水清洗表面的盐，每12h更换一次水，浸泡三天后沥干，自然晒干后粉粹，得到裙带梗粉末；
所述步骤(2)具体按照以下步骤：将裙带梗粉末0.15mol/LCaCl2，料液比1∶15，70℃下提取3h，收集滤液，减压浓缩，得到裙带梗浓缩液；
所述步骤(3)具体按照以下步骤：将裙带梗浓缩液使用80％乙醇醇沉过夜，离心收集沉淀后用少量去离子水复溶，氯化717阴离子树脂55℃脱色12h，Sevage法除蛋白三次以上，直至两相分界处无白色絮状物，3500Da透析48h、冷冻干燥得到粗多糖，氯仿∶正丁醇＝4∶1。
5.根据权利要求1-2任一项所述的一种具有α-葡萄糖苷酶活性的裙带梗多糖UPPS-1在制备糖尿病抑制剂药物中的应用。
6.根据权利要求5所述的应用，其特征在于：所述糖尿病抑制剂药物具有抑制肠黏膜上的α-葡萄糖苷酶，使淀粉分解为葡萄糖的速度减慢，从而减少和延缓小肠对葡萄糖的吸收，改善糖尿病人血糖异常的活性。
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