CN116375902A - 一种提高免疫功能的葡萄渣多糖及其制备方法和应用 - Google Patents
一种提高免疫功能的葡萄渣多糖及其制备方法和应用 Download PDFInfo
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- CN116375902A CN116375902A CN202310400905.7A CN202310400905A CN116375902A CN 116375902 A CN116375902 A CN 116375902A CN 202310400905 A CN202310400905 A CN 202310400905A CN 116375902 A CN116375902 A CN 116375902A
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- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明提供了一种提高免疫功能的葡萄渣多糖及其制备方法和应用，属于生物医学技术领域。本发明首先将葡萄渣通过无水乙醇除脂，随后使用蒸馏水采用300W超声提取40min，得到葡萄渣提取液；之后采用真空旋转蒸发浓缩的方式将葡萄渣提取液浓缩，同时利用乙醇进行醇沉过夜；之后收集醇沉后的沉淀用蒸馏水溶解，用Sevag法除去蛋白，AB‑8型大孔树脂除色素；并且通过滤膜为10Kda的超滤机除去小分子物质，最后真空冷冻干燥得到葡萄渣多糖。本发明所制备的多糖可以显著的降低肝损伤并抑制肝癌细胞增殖，因此有助于实现葡萄渣的变废为宝。
Description
技术领域
本发明属于生物医学技术领域，尤其涉及一种提高免疫功能的葡萄渣多糖及其制备方法和应用。
背景技术
目前，葡萄在全球总产量约为2338万吨，在所有水果中一直位居前五。如今葡萄酒产业已颇具规模，根据国际葡萄酒进出口组织(OIV)的数据。然而，在酿酒过程中也相应产生了大量的固体残渣(约占葡萄重量的25％)。这些主要由葡萄渣组成的固体残渣通常会作为废物排放到环境中，操作不当便会造成废物分解、气味、浸出等更加严重的环境污染。
近年来，植物多糖作为天然产物因其低毒、安全、高效等特点已成为最近几年的研究热点。然而，关于葡萄渣副产物中提取多糖是否具有抗氧化保肝活性研究还没有报道，其提取过程、纯化方式、结构表征和生物活性报道也很少。因此，葡萄渣多糖的商业价值以及其作为抗氧化的潜力值得进一步研究。
发明内容
本发明的目的在于提供一种提高免疫功能的葡萄渣多糖及其制备方法和应用，从而发挥葡萄渣的商业价值。
为实现上述目的，本发明提供了如下技术方案：
本发明提供了一种提高免疫功能的葡萄渣多糖，所述葡萄渣多糖的制备方法包括如下步骤：
(1)将葡萄渣通过无水乙醇浸泡2h除脂，随后按照1:3的料液比加入蒸馏水采用300W超声提取40min，得到葡萄渣提取液；
(2)采用真空旋转蒸发浓缩的方式将葡萄渣提取液浓缩为原体积的五分之一，然后按照样品与乙醇比例为1:4加入无水乙醇在4℃下醇沉过夜；
(3)收集醇沉后的沉淀用蒸馏水溶解，用Sevag法除去蛋白，AB-8型大孔树脂除色素；
(4)通过滤膜为10Kda的超滤机除去小分子物质，最后真空冷冻干燥得到葡萄渣多糖。
优选地，所述葡萄渣多糖降低CCl4诱导的肝组织损伤；所述葡萄渣多糖降低CCl4引起的肝脏炎症；所述葡萄渣多糖降低CCl4诱导的肝组织氧化应激；降低所述葡萄渣多糖提高巨噬细胞的吞噬能力。
其次，本发明提供了葡萄渣多糖在制备降低CCl4诱导的肝组织损伤治疗药物中的应用。
优选地，所述药物可同时降低CCl4导致的肝细胞肿胀坏死和空泡化，炎性细胞浸润，以及血清中ALT和AST的含量增加。
其次，本发明提供了葡萄渣多糖在制备抑制肝癌细胞增殖的药物中的应用，所述葡萄渣多糖由权利要求1所述的制备方法制备得到。
其次，本发明提供了一种用于抑制干细胞细胞增殖的组合药物，所述组合药物的核心有效成分为葡萄渣多糖和甘草甜素；
所述葡萄渣多糖由权利要求1所述的制备方法制备得到；
所述组合药物中葡萄渣多糖的浓度为10-20μg/mL，所述组合药物中甘草甜素的浓度为10-20μg/mL。
其次，本发明提供了葡萄渣多糖在制备降低CCl4引起的肝脏炎症治疗药物中的应用。
优选地，所述药物降低CCl4引起的血液中炎症因子TNF-α和IL-6水平增加。
其次，本发明所提供的葡萄渣多糖在制备降低CCl4引起的肝脏氧化应激药物中的应用。
优选地，所述药物可同时降低肝损伤标志物MDA含量，升高SOD，CAT和GSH酶活性。
其次，本发明提供了葡萄渣多糖在制备提高巨噬细胞吞噬能力的药物中的应用。
本发明的有益效果在于：
通过BBD响应面法优化了葡萄渣多糖的最优提取方式，通过体内动物实验发现葡萄渣多糖能抑制炎症因子TNF-α、IL-6的表达；改善了CCCl4急性肝损伤模型造成的肝脏形态；显著降低了肝脏指数、ALT、AST的活性以及氧化应激标志物MDA的含量，升高了抗氧化酶SOD、CAT、GSH的活性，并提高了巨噬细胞吞噬能力；
除此之外，本发明发现本发明制备的葡萄渣多糖能够有效的肝癌细胞的增殖，并且当其浓度在10-20μg/mL时，能够与甘草甜素产生一个协同的抑制效果，从而可以实现二者在相对较低的浓度即可实现显著的抑制效果，从而节约成本并减少毒副作用。
附图说明
图1为本发明所述料液比对葡萄渣多糖得率的影响示意图；
图2为本发明超声功率对葡萄渣多糖得率的影响示意图；
图3为本发明超声时间对葡萄渣多糖得率的影响示意图；
图4为本发明葡萄渣多糖紫外吸收图谱；
图5为本发明葡萄渣多糖红外吸收图谱；
图6为本发明葡萄渣多糖的SEM；
图7为本发明葡萄渣多糖对肝脏指数的影响；
图8为本发明葡萄渣多糖对肝脏器官的影响；
图9为本发明葡萄渣多糖对TNF-α和IL-6细胞因子分泌的影响；
图10为本发明葡萄渣多糖对ALT和AST酶含量的影响；
图11为本发明葡萄渣多糖对SOD、CAT、MDA和GSH酶活性的影响；
图12为本发明葡萄渣多糖对小鼠腹腔巨噬细胞吞噬能力的影响
具体实施方式
实施例1
筛选葡萄渣多糖的最佳提取步骤
1.不同料液比对多糖提取的影响
(1)将葡萄渣通过无水乙醇浸泡2h除脂，按照1:1；1:2，1:3，1:4，1:5的料液加入蒸馏水采用300W超声提取40min，得到葡萄渣提取液；
(2)采用真空旋转蒸发浓缩的方式将葡萄渣提取液浓缩为原体积的五分之一，然后按照样品与乙醇比例为1:4加入无水乙醇在4℃下醇沉过夜；
(3)收集醇沉后的沉淀用蒸馏水溶解，用Sevag法(正丁醇:氯仿1:4)除去蛋白，AB-8型大孔树脂除色素；
(4)通过超滤机滤膜为10Kda除去小分子物质，最后真空冷冻干燥得到葡萄渣多糖。
(5)以葡萄糖为标样，用苯酚-硫酸法测定粗多糖含量。标准曲线回归方程为Y＝0.4312x+0.0625(x为葡萄糖质量浓度，范围为20～120mg/L，Y为吸光度)，R＝0.9978，根据方程可计算粗多糖含量。
2.不同提取功率对于多糖提取的影响
考察不同提取功率100W、200W、300W、400W、500W，每组平行重复三次，并计算产率。其他操作与料液比对葡萄渣多糖提取的影响的操作相同，最后冷冻干燥并计算多糖提取率。
3.不同提取时间对于多糖提取的影响
考察不同提取时间为10、20、30、40、50min，每组平行重复三次，并计算产率。其他操作与料液比对葡萄渣多糖提取的影响的操作相同，最后冷冻干燥并计算多糖提取率
在单因素试验的基础上，采用Box－Benhnken的中心组合设计对超声辅助葡萄渣多糖工艺进行优化，响应面优化实验因素和水平表见表1。根据响应面优化设计实验结果进行验证实验，并比较预测多糖得率与实际多糖得率。
表1响应面水平实验设计
在单因素试验结果的基础上利用Box－Behnken Design(BBD)中心组合设计，以葡萄渣多糖提取得率Y(％)为响应值，考察三个独立变量:料液比(A)、超声功率(B)和提取时间(C)(每个因素取3个水平)对提取得率的影响。响应面试验优化葡萄渣多糖提取条件的结果如表2所示，所有试验均进行3次；并利用Design Expert(8.0版)软件进行数据分析。采用ANOVA法进行方差分析。在P＜0.05时有统计学意义。
料液比对超声波辅助葡萄渣多糖得率的影响见图1。由图1可知：随着料液比的增加，葡萄渣多糖得率逐渐升高，当料液比为1:3时，多糖得率最高。这是因为，当料液比较低时，溶剂与样品接触不充分从而导致提取不彻底。随着料液比的继续增加，多糖的得率呈下降趋势，这可能是因为随着料液比的增加使随后的料液的分离浓缩操作难度增加，导致多糖的损失增加。因此，最优料液比为1:3。
超声功率对超声波辅助葡萄渣多糖得率的影响见图2。由图2可知，随着超声功率的增加，葡萄渣多糖得率逐渐升高，当超声功率为300W时，多糖得率最高，这是因为随着超声波产生的空穴效应和震动利于多糖的溶解。随着超声功率的继续增加，多糖得率呈下降趋势，这是因为过高的超声波功率会导致多糖的降解,因此最优的超声功率为300W。
超声时间对超声波辅助葡萄渣多糖得率的影响见图3。由图3可知：随着超声时间的增加，葡萄渣多糖得率逐渐升高，当超声时间为440min时，鲜食葡40min时，多糖已经基本溶出，随着超声时间的增加，不会提高葡萄渣多糖的得率，反而使部分小分子多糖降解，导致得率下降。因此最优的超声时间为40min。
表2响应面实验设计及结果
回归方程拟合及方差分析
对葡萄渣多糖得率的响应面实验结果进行回归分析，结果见表(2)。对三个因素进行回归拟合后得到回归方程：
Y＝8.14+0.12*A-0.071*B-0.039*C-0.023*AB-2.500E-003*AC-0.055*BC-1.76*A2-0.46*B2-0.71*C2
由方差分析可知，校正系数R2＝0.9991,R2模型的P值小于0.0001，为极显著；失拟项P值大于0.05，不显著。这说明模型拟合度较好，说明实验的二次模型拟合度较高，可正确反映个因素与响应值之间的变化关系。由模型的方差分析可得出料液比(A)、超声功率(B)、超声时间(C)、、料液比与超声功率交互项(AB)、料液比与料超声时间交互项(AC)、超声功率与超声时间交互项(BC)及个因素的二次项对响应值有显著影响。各因素对响应值的影响显著性排序为A＞C＞B。
由响应面和等高线图可知，各因素之间的响应面均较陡峭，说明各因素之间的交互作用均较强。根据所得到的模型，预测最优工艺条件为提取时间为料液比为1:3.03，提取功率为292.47W，提取时间为39.75min。
经Design-Expert8.0.6预测模型极值点，结果显示最佳提取条件为料液比为1:3.03，提取功率为292.47W，提取时间为39.75min。根据实际操作设计验证实验，其提取条件为：料液比为1:3，提取功率为300W，提取时间为40min。，取条件进行3次平行实验，实验结果为在次条件下的多糖得率为8.149％，验证实验结果与预测值接近，表明该模型拟合度良好，对此提取条件取葡萄渣多糖的工艺优化合理、有效。
实施例2
检测所提取的葡萄渣多糖的性质
(1)采用紫外分光光度计测量260-280nm处的吸收峰、红外吸收图谱和SEM；
(2)检测得到的结果被展示在图4、图5和图6中。
如图4所示，葡萄渣多糖(GPP)在260nm和280nm处没有吸收峰，说明GPP不含蛋白质或核酸，说明本发明得到成分均匀并且不含杂质的GPP。
如图5所示，FT-IR图谱显示了500-4000cm-1之间的吸收峰。在3390、2930cm-1处的强吸收带分别代表糖分子内羟基O-H和C-H伸缩振动，这两条为多糖的特征峰。在161cm-1附近检测到的峰是由于羧基COO-的伸缩振动，这表明多糖中含有糖醛酸，1380cm-1处的峰值可能归因于C-H弯曲振动，800-1200cm-1之间的区域被认为是指纹区域，因为它能够反映糖环的振动。在1060和1100cm-1处观察到的两个吸收峰于糖苷键C-O-C拉伸有关，这表明可能存在吡喃糖环。此外，在840cm-1和816cm-1处的吸收表明存在α型糖苷键。FT-IR结果表明，GPP具有典型的多糖结构，说明本实验方法获得的提取物均为多糖类物质。
如图6所示在400×下，GPP具有不规则团状聚集存在，表面带有细小、粘合的凸颗粒。在2000×倍镜下观察到团状之间孔洞较多，这可能是由于分子间吸引力的相互排斥所致。
实施例3
葡萄渣多糖对CCl4小鼠急性肝损伤肝脏指数影响以及肝脏组织的病理学观察
(1)将30只SPF级ICR小鼠随机分为5组，每组6只，阴性雌雄各半，体重20±2g，包括正常组、模型组，每日灌胃生理盐水；阳性对照组(水飞蓟素治疗组)，每天灌胃水飞蓟素溶液(100mg/kg)；两个GPP组：低剂量组(GPP-L)和高剂量组(GPP-H)，连续14天每日灌胃不同浓度摩尔多瓦葡萄多糖溶液(100、200mg/kg)。
(2)第15天，除空白组外，所有小鼠腹腔注射含0.1％ CCl44橄榄油溶液，而空白组小鼠腹腔注射相同体积的不含CCl4的橄榄油。
(3)通过分析天平称量每只小鼠重量，处死后立即测量肝脏重量。
(4)肝脏组织保存再10％多聚甲醛里，进行HE染色切片，在显微镜下观察拍照。
肝脏指数如图7所示，与正常组相比，CCl4模型组的小鼠肝脏肿大。低剂量组肝脏指数降低；高剂量组和阳性对照组以及正常组无较大差异。
肝脏病理学组织实验结果如图8所示，对照组肝脏结构正常，肝细胞排列整齐，细胞结构清晰。然而，在CCl4的肝组织中观察到严重的损伤模型包括肝细胞大面积肿胀坏死，空泡化，炎性细胞浸润等形态组织变化。相比之下，水飞蓟素组和GPP组小鼠肝细胞空泡区域减小，肝细胞肿胀变性，炎性浸润等病理现象明显改善。GPP处理组肝细胞损伤均有不同程度的明显减轻。上述结果表明GPP能一定程度上保护肝脏减轻CCL4诱导的肝组织损伤，且浓度越高改善效果更好。
实施例4
葡萄渣多糖对炎症因子TNF-α和IL-6的影响
为了验证GPP对CCL4诱导小鼠肝损伤模型中炎症因子水平的影响，本发明测定了参与正常炎症反应和免疫反应的重要炎症相关指标TNF-α和IL-6。
(1)将小鼠从眼窝收集血液到无菌管中，在3000r/min和4℃下离心10min以提取上清液血清并将其储存在-80℃。
(2)根据制造商说明，使用上海酶联生物科技有限公司的酶联免疫吸附试验(ELISA)试剂盒测量血清TNF-α和IL-6的含量。
如图9所示，与正常组相比，模型组的TNF-α和IL-6水平显著增加(p<0.05)，表明造模成功；然而与模型组相比GPP预处理后显著降低了TNF-α和IL-6水平，GPP-H组与阳性对照组表达相似，说明高剂量的GPP能缓解CCl4引起的肝脏炎症。
实施例5
葡萄渣多糖对血清ALT和AST的影响
血清ALT和AST的酶活性是肝损伤的敏感指标，因此，本发明对其进行了检测
(1)将小鼠从眼窝收集血液到无菌管中，在3000r/min和4℃下离心10min以提取上清液血清并将其储存在-80℃。
(2)根据制造商说明，使用上海酶联生物科技有限公司的酶联免疫吸附试验(ELISA)试剂盒测量血清ALT和AST的含量。
如图10所示，与正常对照组相比，CCL4处理的小鼠血清ALT和AST活性分别显著增加；然而，与模型组相比，水飞蓟素组显著降低ALT和AST酶含量，观察发现100mg/ml和200mg/ml剂量的GPP组分别以剂量依赖性有效地防止了CCL4诱导血清ALT和AST升高。结果表明GPP对CCl4诱导的小鼠肝组织损伤有保护作用。
实施例6
葡萄渣多糖对肝脏抗氧化酶活性的影响
本发明通过SOD、CAT、MDA和GSH水平来评估CCl4诱导的小鼠急性肝损伤中的氧化应激。
(1)小鼠处死后，一半肝脏加入9倍体积的PBS溶液研磨肝组织，以5000r/min rpm和4℃下离心15min收集上清液，储存在-80℃以供进一步分析。
(2)根据制造商说明，使用上海酶联生物科技有限公司的酶联免疫吸附试验(ELISA)试剂盒测量肝组织匀浆中MDA，SOD，CAT和GSH的活性。
如图11所示，与正常组相比，模型组MDA活性升高，SOD，CAT和GSH酶活性显著性降低(P<0.001)，表明CCl4诱导产生的肝损伤造模成功。然而，GPP预处理组的小鼠以浓度依赖性方式显著改善。结果表明，GPP具有很强的抑制CCl4引起的肝组织氧化损伤能力。
实施例7
葡萄渣多糖对小鼠腹腔巨噬细胞吞噬能力的影响
(1)在给药前3天，每天给小鼠腹腔注射6％淀粉肉汤溶液1mL。最后一次灌胃后，禁食24h，给每只小鼠腹腔注射5％已制备的鸡红细胞悬液1mL进行免疫，对小鼠腹部充分按摩其在腹腔内充分分散。
(2)注射30min后脱颈处死小鼠，腹部注入2mL生理盐水稀释后抽取1mL腹腔液。将腹腔液滴于载玻片上进行瑞氏染色在显微镜下观察计数。吞噬率计算方法：吞噬率＝吞噬鸡红细胞的巨噬细胞数/巨噬细胞总数×100％。
如图12所示，与空白对照组小鼠相比，CTX环磷酰胺组小鼠腹腔巨噬细胞吞噬率显著降低(P<0.001)，葡萄渣多糖给药后，均显著增高巨噬细胞吞噬率，且随浓度的升高而呈浓度依赖性。
实施例8
(1)取对数期生长的HepG2细胞在RPMI1640培养基中，置于37℃，体积分数5％的CO2培养箱中培养过夜。待细胞贴壁后，用胰酶消化进行传代接种于96孔板中，24h后给予不同处理，分为对照组、甘草甜素组、葡萄渣多糖组、葡萄渣多糖+甘草甜素组；
(2)用RPMI1640培养基将药物倍比稀释至一系列浓度，葡萄渣多糖组浓度为1μg/mL、5μg/mL、10μg/mL和20μg/mL；甘草甜素组浓度为1μg/mL、5μg/mL、10μg/mL和20μg/mL，葡萄渣多糖+甘草甜素浓度为1+1μg/mL、5+5μg/mL、10+10μg/mL和20+20μg/mL，对照组为不加药处理的正常细胞。
(3)吸出96孔板中的培养基，再加入100含有药物的培养基，每个药物浓度设置6个复孔。加药处理24h后，吸出药物培养基，加入含有10％ CCK-8的RPMI1640培养基置于培养箱中孵育30min后，在波长为450nm处用酶标仪检测其吸光度。
(4)计算HepG2细胞的增殖抑制率：增殖抑制率＝1-A实验/A对照；采用金氏公式计算药物联用效果：q＝Ea+b/(Ea+Eb-Ea*Eb).注：q为增效指数；Ea+b为实测合并效应；Ea、Eb分别为A、B单用的效应；0.85<q<1.15为相加(+)，1.15<q<20为增强(++)，0.55<q<0.85为拮抗,实验结果如表1和表2所示。
表1甘草甜素、葡萄渣多糖和甘草甜素+葡萄渣多糖酸不同培养时间对HepG-2细胞增殖抑制率
表2甘草甜素+葡萄渣多糖酸不同培养时间的q值
从表1和表2可以看出，葡萄渣多糖能够有效的抑制肝癌细胞HepG2的增殖，并且当其浓度为10-20μg/mL时，其能够与甘草甜素产生协同效果来抑制肝癌细胞增殖。
Claims (9)
1.一种提高免疫功能的葡萄渣多糖，其特征在于，所述葡萄渣多糖的制备方法包括如下步骤：
(1)将葡萄渣通过无水乙醇浸泡2h除脂，随后按照1:3的料液比加入蒸馏水采用300W超声提取40min，得到葡萄渣提取液；
(2)采用真空旋转蒸发浓缩的方式将葡萄渣提取液浓缩为原体积的五分之一，然后按照样品与乙醇比例为1:4加入无水乙醇在4℃下醇沉过夜；
(3)收集醇沉后的沉淀用蒸馏水溶解，用Sevag法除去蛋白，AB-8型大孔树脂除色素；
(4)通过滤膜为10Kda的超滤机除去小分子物质，最后真空冷冻干燥得到葡萄渣多糖。
2.根据权利要求1所述的葡萄渣多糖，其特征在于，所述葡萄渣多糖降低CCl4诱导的肝组织损伤；所述葡萄渣多糖降低CCl4引起的肝脏炎症；所述葡萄渣多糖降低CCl4诱导的肝组织氧化应激；降低所述葡萄渣多糖提高巨噬细胞的吞噬能力。
3.如权利要求1所述的葡萄渣多糖在制备降低CCl4诱导的肝组织损伤治疗药物中的应用。
4.根据权利要求3所述的应用，其特征在于，所述药物可同时降低CCl4导致的肝细胞肿胀坏死和空泡化，炎性细胞浸润，以及血清中ALT和AST的含量增加。
5.如权利要求1所述的葡萄渣多糖在制备抑制肝癌细胞增殖的药物中的应用，其特征在于，所述葡萄渣多糖由权利要求1所述的制备方法制备得到。
6.一种用于抑制干细胞细胞增殖的组合药物，其特征在于，所述组合药物的核心有效成分为葡萄渣多糖和甘草甜素；
所述葡萄渣多糖由权利要求1所述的制备方法制备得到；
所述组合药物中葡萄渣多糖的浓度为10-20μg/mL，所述组合药物中甘草甜素的浓度为10-20μg/mL。
7.如权利要求1所述的葡萄渣多糖在制备降低CCl4引起的肝脏氧化应激药物中的应用。
8.根据权利要求7所述的应用，其特征在于，所述药物可同时降低肝损伤标志物MDA含量，升高SOD，CAT和GSH酶活性。
9.如权利要求1所述的葡萄渣多糖在制备提高巨噬细胞吞噬能力的药物中的应用。
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