CN113116920A - 枸杞多糖在制备抗疲劳药物、保健品或食品中的应用 - Google Patents
枸杞多糖在制备抗疲劳药物、保健品或食品中的应用 Download PDFInfo
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Abstract
本发明公开了枸杞多糖在制备抗疲劳药物、保健品或食品中的应用。分子量为92.4kDa的枸杞多糖能够明显增加肝糖原和肌糖原储备，提高超氧化物歧化酶活力，显著降低血清尿素氮、血乳酸、丙二醛水平，相较于其他分子量的枸杞多糖而言，表现出更为优异的抗疲劳作用，适用于制备抗疲劳药物、保健品或食品。
Description
技术领域
本发明属于药物技术领域，涉及枸杞多糖在制备抗疲劳药物、保健品或食品中的应用。
背景技术
疲劳是机体在从事脑力活动或体力活动时由于消耗过大，达到一定程度时出现的一种正常的生理现象。疲劳通常会导致一系列继发性问题，例如抑郁和焦虑，同时还与认知障碍、睡眠质量差、身体机能障碍和能量失衡有关。长期疲劳会增加罹患癌症、多发性硬化症、人类免疫缺陷病毒感染和帕金森氏病的风险。消除和缓解疲劳一直以来备受运动医学、军事医学和航天医学等领域的关注。疲劳是一种复杂而普遍的生理现象，没有明确的病因，可能需要长期服药，而长期用药可能会导致一些不良反应或毒副作用。因此，找到具有明确功效且较少副作用的抗疲劳潜在药物或制剂是目前关注的重点。
枸杞为茄科植物枸杞的成熟果实，广泛分布于中国西北部、欧洲东南部和地中海干旱半干旱地区。从枸杞提取物中分离得到的枸杞多糖(L.barbarum polysaccharide，LBP)是枸杞中含量最丰富的成分之一，占干果的5％～8％，被认为是发挥生物学功效的重要组成成分。枸杞多糖因具有抗衰老、抗氧化、抗糖尿病、抗癌、细胞保护、神经保护和免疫调节等保健作用，常被作为药物和功能性食品使用。然而天然多糖分子量与其在体内的吸收和运输密切相关，从而影响生物活性。
发明内容
本发明的目的在于提供一种具有特定分子量的枸杞多糖在制备抗疲劳药物、保健品或食品中的应用。
本发明中，所述的枸杞多糖的分子量为92.4kDa。
本发明中，所述的枸杞多糖通过以下步骤制备：
(1)将枸杞粉末在80℃下用95％乙醇回流脱脂，重复数次，收集滤渣并干燥；
(2)将干燥后的滤渣按照料液比1:30加入蒸馏水，在90℃下提取，重复数次，5℃下离心收集上清液，浓缩，加入95％乙醇分散，4℃下静置过夜，5℃下离心收集沉淀；
(3)将沉淀用蒸馏水溶解，再按体积比5:1加入Sevage试剂，充分振荡，然后在5℃下离心收集上清液，重复操作至中间无变性蛋白质层出现；
(4)将上清液减压浓缩蒸发除去残留的Sevage试剂，并在4℃下透析，将截留液冷冻干燥获得粗制枸杞多糖：
(5)然后称取粗制枸杞多糖溶于蒸馏水中，缓慢加入无水乙醇，使最终溶液的乙醇浓度为30％，于4℃静置过夜，然后5℃下离心，收集沉淀，冷冻干燥得到30％的醇沉组分LBP-Ⅰ；
(6)将LBP-Ⅰ溶于蒸馏水中，经葡聚糖凝胶Sephadex G-100柱层析纯化，用蒸馏水进行洗脱，收集洗脱液，通过苯酚-硫酸法跟踪监测多糖，合并馏分，浓缩并冻干，得到纯化的分子量为92.4kDa的枸杞多糖。
优选地，步骤(1)中，脱脂时间为2h，重复次数为3次。
优选地，步骤(2)具体为：将干燥后的滤渣按照料液比1:30加入蒸馏水，在90℃下提取2h，重复2次，4000r/min、5℃下离心10min，合并上清液，浓缩至原来体积的四分之一，加入4倍体积的95％乙醇，4℃下静置过夜，4000r/min、5℃下离心10min，收集沉淀。
优选地，步骤(3)中，振荡时间为20min，离心转速为4000r/min，离心时间为10min。
优选地，步骤(4)中，透析袋的截留分子量为3500Da，透析时间为48h。
优选地，步骤(5)中，离心转速为8000r/min，离心时间为15min。
优选地，步骤(6)中，蒸馏水的洗脱速度为0.3mL/min。
本发明中，所述的药物、保健品或食品为缓解运动疲劳的药物、保健品或食品。
进一步地，所述的药物是血清尿素氮(BUN)、乳酸(BLA)和丙二醛(MDA)含量，提高血糖、超氧化物歧化酶(SOD)水平的药物、保健品或食品。
本发明首次发现不同分子量的枸杞多糖的抗疲劳提升作用存在明显差异，其中分子量为92.4kDa的抗疲劳提升效果最为显著，可以显著延长小鼠负重游泳时间，有效降低疲劳小鼠BLA和BUN以及MDA含量，提高SOD、肝糖原和肌糖原水平，与其他分子量的枸杞多糖相比较，表现出更为优异的抗疲劳作用。
附图说明
图1为不同分子量枸杞多糖对小鼠肝糖原(A)和肌糖原(B)的影响。其中，*表示存在显著性差异(p<0.05)；**表示存在极显著性差异(p<0.01)。
图2为不同分子量枸杞多糖对小鼠BUN(A)、BLA(B)、MDA(C)和SOD(D)水平的影响。其中，*表示存在显著性差异(p<0.05)；**表示存在极显著性差异(p<0.01)。
具体实施方式
下面结合具体实施例和附图对本发明做进一步详细描述。
实施例1
1.实验材料
1.1材料与试剂
糖原、尿素氮(BUN)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)以及丙二醛(MDA)测定试剂盒(南京建成生物工程研究所)；枸杞子(甘肃省定西市漳县金钟镇三农中药材种植农民专业合作社)、西洋参口服液(每100mL含：总皂甙(以人参皂甙Re计)230mg，福建省力菲克药业有限公司)
1.2实验仪器
紫外可见分光光度计(Lambda 35，美国珀金埃尔默股份有限公司)；多功能酶标仪(Tecan infinite 200pro，Tecan Austria GmbH公司)
1.3实验动物
雄性ICR小鼠(20±2g)购自扬州大学比较医学中心，动物合格证号为SCXK(苏)2017-0007。使小鼠在25℃的环境中适应一周，每天光/暗(12/12h)循环，并提供足够的饲料和饮用水
2.实验方法
2.1枸杞多糖的制备
将枸杞粉末在80℃下用95％乙醇回流脱脂2h，重复3次，收集滤渣并干燥。称取干燥后的滤渣按照料液比1:30加入蒸馏水，在90℃下提取2h，重复2次，4000r/min、5℃下离心10min，合并上清液，浓缩至原来体积的四分之一，加入4倍体积的95％乙醇，4℃下静置过夜，4000r/min、5℃下离心10min，收集沉淀。将所得沉淀用蒸馏水溶解，再按体积比5:1加入Sevage试剂，充分振荡20min，在4000r/min、5℃下离心10min后收集上清液，重复操作至中间无变性蛋白质层出现。将上清液减压浓缩蒸发除去残留的Sevage试剂，并在4℃下透析48h(Mw 3500Da)，将截留液冷冻干燥获得粗制枸杞多糖(LBP)。然后称取LBP溶于蒸馏水中，缓缓加入无水乙醇，使最终溶液的乙醇浓度为30％，于4℃静置过夜，然后离心(8000r/min，15min，5℃)，收集沉淀，冷冻干燥得到30％的醇沉组分LBP-Ⅰ；在上清液中继续缓缓加入乙醇，使最终溶液的乙醇浓度为50％，4℃下静置过夜后，离心(8000r/min，15min，5℃)，收集沉淀，冷冻干燥得到50％的醇沉组分LBP-Ⅱ；按相同的操作，使最终溶液的乙醇浓度为70％时，得到70％的醇沉组分LBP-Ⅲ。最后称取LBP-Ⅰ、LBP-Ⅱ和LBP-Ⅲ各40mg溶于5mL蒸馏水中，经葡聚糖凝胶Sephadex G-100柱(1.5cm×60cm)层析纯化，用蒸馏水以0.3mL/min速度进行洗脱，自动收集每管3mL。通过苯酚-硫酸法跟踪监测多糖，合并馏分，浓缩并冻干，得纯化多糖LBP-Ⅰ-1、LBP-Ⅱ-1和LBP-Ⅲ-1，命名为LBP1、LBP2和LBP3。
LBP1、LBP2和LBP3纯度分别为80.1％、87.3％和73.4％，分子量分别为92,441Da、7714Da和3188Da。采用红外光谱法、气相色谱法、1H NMR谱和35C NMR谱等方法对多糖结构进行表征。结果表明3种多糖结构基本相似，均不含有β构型的糖残基。LBP1由阿拉伯糖(24.0％)、木糖(25.0％)、葡萄糖(20.2％)和半乳糖(23.1％)组成；LBP2由阿拉伯糖(28.3％)、木糖(17.8％)、葡萄糖(13.4％)和半乳糖(23.4％)组成；LBP3由阿拉伯糖(26.3％)和葡萄糖(30.3％)组成。
2.2实验分组
将小鼠随机分成10组：空白组(给予等体积生理盐水)、阳性对照组(给予西洋参口服液，剂量参考说明书中推荐每日摄入量为6.7mg/kg/d)、LBP1、LBP2和LBP3的低、中、高剂量组(低、中、高剂量组分别灌胃5、10、20mg/kg/d枸杞多糖溶液)，每组20只，连续灌胃30天。
2.3测定负重游泳时间
最后一次给药1h后，从每组中随机选取10只小鼠并按体重的7％在尾部附加铅片置于游泳箱(50cm×50cm×40cm)中，游泳箱中水深不低于30cm，水温维持在30±1℃。以小鼠头部沉没水中8s以上作为试验终点，记录开始游泳至力竭的时间，为负重力竭游泳时间。
2.4测定肝糖原、肌糖原、BUN、BLA、SOD和MDA水平
每组中另外10只小鼠进行无负重游泳30min，结束后立即采用眼眶取血，室温静置15min使血液凝固，并以3000r/min，4℃离心10min收集血清，置于冰箱中冷藏备用，按照试剂盒中的方法测定血清中BUN、BLA、SOD和MDA水平。同时，收集肝脏和后腿肌肉，生理盐水漂洗后，用滤纸吸干，分别称取肝脏100mg，肌肉500mg置于试管中，按照试剂盒中的方法分析肝糖原和肌糖原含量。
2.5统计学处理
实验所得数据采用平均值±标准偏差(SD)表示，使用SPSS 23.0软件进行单因素方差分析多重比较，p<0.05表示具有显著性差异，p<0.01表示具有极显著差异。
3.实验结果
3.1不同分子量枸杞多糖对小鼠负重游泳时间的影响
如表1所示，经过30天的灌胃各组小鼠体征正常，未出现死亡情况，体重正常增长。与空白组相比，灌胃不同剂量的LBP1、LBP2和LBP3小鼠负重游泳时间均有所延长，说明不同分子量的枸杞多糖均能够有效缓解小鼠运动疲劳。其中，高剂量的LBP1组中小鼠负重游泳时间较阳性对照组有所提升，但不存在显著性差异。当灌胃剂量相同时，LBP1实验组小鼠负重游泳时间较LBP2和LBP3两组有所延长，其中高剂量的LBP1组游泳时间显著长于高剂量的LBP3。说明LBP1对延长小鼠负重游泳时间的效果最为显著，更能够提高其抗疲劳能力。
表1
3.2不同分子量枸杞多糖对小鼠肝糖原、肌糖原含量的影响
图1显示了各组小鼠在不负重游泳30min之后肝糖原和肌糖原的含量。结果表明LBP1和LBP2各剂量组中肝糖原和肌糖原含量均极显著高于空白组(p<0.01)，表明LBP1和LBP2具有增强糖原储备，为身体提供能量，缓解身体疲劳和增强运动耐力的能力。
3.3不同分子量枸杞多糖对小鼠BUN、BLA、SOD和MDA水平的影响
图2显示了各组小鼠不负重游泳30min后血液中SOD活性以及BUN、BLA和MDA的水平。结果显示，经不同分子量的枸杞多糖治疗后BUN、BLA和MDA含量较空白组明显降低，同时SOD活力显著提高，并且LBP1组治疗效果更明显。
综上所述，经小鼠游泳模型发现，不同分子量的枸杞多糖均能有效缓解运动疲劳症状，特别是分子量为92.4kDa的LBP1，能够显著延长小鼠负重游泳时间，并对运动后小鼠血液生化指标有改善作用，具有良好的抗疲劳作用。
Claims (10)
1.枸杞多糖在制备抗疲劳药物、保健品或食品中的应用，其特征在于，所述的枸杞多糖的分子量为92.4kDa。
2.根据权利要求1所述的应用，其特征在于，所述的枸杞多糖通过以下步骤制备：
(1)将枸杞粉末在80℃下用95％乙醇回流脱脂，重复数次，收集滤渣并干燥；
(2)将干燥后的滤渣按照料液比1:30加入蒸馏水，在90℃下提取，重复数次，5℃下离心收集上清液，浓缩，加入95％乙醇分散，4℃下静置过夜，5℃下离心收集沉淀；
(3)将沉淀用蒸馏水溶解，再按体积比5:1加入Sevage试剂，充分振荡，然后在5℃下离心收集上清液，重复操作至中间无变性蛋白质层出现；
(4)将上清液减压浓缩蒸发除去残留的Sevage试剂，并在4℃下透析，将截留液冷冻干燥获得粗制枸杞多糖：
(5)然后称取粗制枸杞多糖溶于蒸馏水中，缓慢加入无水乙醇，使最终溶液的乙醇浓度为30％，于4℃静置过夜，然后5℃下离心，收集沉淀，冷冻干燥得到30％的醇沉组分LBP-Ⅰ；
(6)将LBP-Ⅰ溶于蒸馏水中，经葡聚糖凝胶Sephadex G-100柱层析纯化，用蒸馏水进行洗脱，收集洗脱液，通过苯酚-硫酸法跟踪监测多糖，合并馏分，浓缩并冻干，得到纯化的分子量为92.4kDa的枸杞多糖。
3.根据权利要求2所述的应用，其特征在于，步骤(1)中，脱脂时间为2h，重复次数为3次。
4.根据权利要求2所述的应用，其特征在于，步骤(2)具体为：将干燥后的滤渣按照料液比1:30加入蒸馏水，在90℃下提取2h，重复2次，4000r/min、5℃下离心10min，合并上清液，浓缩至原来体积的四分之一，加入4倍体积的95％乙醇，4℃下静置过夜，4000r/min、5℃下离心10min，收集沉淀。
5.根据权利要求2所述的应用，其特征在于，步骤(3)中，振荡时间为20min，离心转速为4000r/min，离心时间为10min。
6.根据权利要求2所述的应用，其特征在于，步骤(4)中，透析袋的截留分子量为3500Da，透析时间为48h。
7.根据权利要求2所述的应用，其特征在于，步骤(5)中，离心转速为8000r/min，离心时间为15min。
8.根据权利要求2所述的应用，其特征在于，步骤(6)中，蒸馏水的洗脱速度为0.3mL/min。
9.根据权利要求1所述的应用，其特征在于，所述的药物、保健品或食品为缓解运动疲劳的药物、保健品或食品。
10.根据权利要求1所述的应用，其特征在于，所述的药物、保健品或食品为降低血清尿素氮、乳酸和丙二醛含量，提高血糖、超氧化物歧化酶水平的药物、保健品或食品。
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| CN114470174A (zh) * | 2022-03-09 | 2022-05-13 | 扬州大学 | 预防及治疗骨质疏松的组合物 |
| CN116195700A (zh) * | 2023-02-01 | 2023-06-02 | 青海大学 | 一种抗疲劳的枸杞多糖泡腾片、制备方法及应用 |
| CN118286169A (zh) * | 2024-04-10 | 2024-07-05 | 青海大学 | 一种缓解慢性疲劳综合征的枸杞多糖咀嚼片 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114470174A (zh) * | 2022-03-09 | 2022-05-13 | 扬州大学 | 预防及治疗骨质疏松的组合物 |
| CN114470174B (zh) * | 2022-03-09 | 2023-11-10 | 扬州大学 | 预防及治疗骨质疏松的组合物 |
| CN116195700A (zh) * | 2023-02-01 | 2023-06-02 | 青海大学 | 一种抗疲劳的枸杞多糖泡腾片、制备方法及应用 |
| CN118286169A (zh) * | 2024-04-10 | 2024-07-05 | 青海大学 | 一种缓解慢性疲劳综合征的枸杞多糖咀嚼片 |
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