CN114456218A - 一种蓝莓花色苷及其制备方法和在制备抗丙烯酰胺神经毒性的功能食品中的应用 - Google Patents
一种蓝莓花色苷及其制备方法和在制备抗丙烯酰胺神经毒性的功能食品中的应用 Download PDFInfo
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Abstract
本发明提供了一种蓝莓花色苷及其制备方法和在制备抗丙烯酰胺神经毒性的功能食品中的应用，属于生物医药技术领域。本发明中天然活性成分蓝莓花色苷有效改善了丙烯酰胺诱导的大鼠体重、摄食和饮水减少及运动障碍；并通过缓解海马和大脑皮层氧化应激和神经炎症，以减少神经元坏死和突触功能损伤，说明本发明能从多个方面改善丙烯酰胺神经毒性。本发明提供了一种安全、有效、易获取的抗丙烯酰胺神经毒性的治疗方案，也为蓝莓花色苷发掘了新的医疗用途。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种蓝莓花色苷及其制备方法和在制备抗丙烯酰胺神经毒性的功能食品中的应用。
背景技术
丙烯酰胺是一种无色无味的水溶性乙烯基单体。丙烯酰胺主要作为聚丙烯酰胺的前体，被广泛应用于废水处理、矿石加工、化妆品制造以及分子生物学实验中。此外，在薯片、薯条、咖啡、饼干、面包等焙烤或油炸食品制作过程中，丙烯酰胺会作为美拉德反应的副产物而生成。体内外及流行病学实验表明，丙烯酰胺暴露可通过诱发氧化应激和神经炎症以诱发机体神经系统功能紊乱，其代表症状包括步态异常、共济失调、体重减轻、精神萎靡、感觉障碍等。此外，新近研究发现，丙烯酰胺可攻击大脑中负责认知等高级神经活动的海马体和前额叶皮层，以引发神经元坏死及突触功能障碍，进而损伤学习和记忆能力。由于丙烯酰胺暴露在生活中几乎无法避免且高度威胁人体健康，因此安全的、有针对性的、易获取的药物或功能食品的开发对全民健康具有重要意义。
花色苷是一种具有良好抗氧化和抗炎性的天然黄酮类物质，其大量存在于蓝莓中。蓝莓花色苷能跨越血脑屏障，对脂多糖、d-半乳糖、甲基汞等多种毒素诱发的神经元和突触损伤有保护作用，以改善认知功能。此外，蓝莓花色苷还能在肝脏和肾脏中抑制丙烯酰胺引发的氧化应激，并促进其脱毒转化。由于其显著的神经保护功能和易于获取的特性，因此应用蓝莓花色苷制备抗丙烯酰胺诱发的神经毒性的功能食品具有重要社会意义。
发明内容
本发明目的是提供一种蓝莓花色苷的医药新用途，即蓝莓花色苷在制备抗丙烯酰胺神经毒性的功能食品中的应用。
本发明提供了一种蓝莓花色苷在制备抗丙烯酰胺神经毒性的功能食品中的应用，所述神经毒性发生在海马和/或大脑皮层。
优选的，所述神经毒性包括海马和/或大脑皮层的神经元和突触损伤、氧化应激和神经炎症中的一种或几种。
本发明还提供了一种所述的蓝莓花色苷的制备方法，包括如下制备步骤：
(1)将蓝莓果实用提取剂提取，得到总提取物；
(2)所述总提取物经固液分离，得到上清液；
(3)浓缩所述上清液得到沉淀；
(4)所述沉淀加水配成溶液，经纯化、干燥后，得到蓝莓花色苷。
优选的，所述提取剂为甲醇、丙酮、水和甲酸按照体积比35～45：35～45： 15～25：0.05～1.5的混合物；所述蓝莓果实与提取剂的质量体积比为25～35g： 110～140mL。
优选的，所述提取为均质提取，所述均质提取的条件为100～200bar， 2～4×2～4min。
优选的，所述纯化的方法为：用Sep-Pak C18柱分别以盐酸溶液、乙酸乙酯洗涤，然后用酸化甲醇洗脱；所述盐酸溶液的浓度为0.008～0.012％，用量为1.8～2.2倍柱体积；所述乙酸乙酯用量为1.8～2.2倍柱体积；所述酸化甲醇为含0.008～0.012％盐酸的甲醇溶液，用量为1.8～2.2倍柱体积。
本发明还提供了一种蓝莓花色苷，所述蓝莓花色苷包括如下重量份的成分：矢车菊素-3-葡萄糖苷85～95份、矢车菊素-3，5-二葡萄糖苷1～10份、芍药色素-3-葡萄糖苷1～10份。
本发明还提供了一种所述的蓝莓花色苷制备的功能食品，所述功能食品中所述蓝莓花色苷的含量为240～260mg/g。
优选的，所述功能食品中还包括药学或食品生产可接受的载体或添加剂；所述药学或食品生产可接受的载体或添加剂包括：矫味剂、填充剂、润滑剂、粘合剂。
优选的，所述矫味剂包括香精和/或甜味剂；所述填充剂为淀粉、糊精和纤维素中的一种或几种；所述润滑剂为硬脂酸镁、滑石粉和微粉硅胶中的一种或几种；所述粘合剂为淀粉浆、聚维酮和纤维素中的一种或几种。
优选的，所述功能食品可制备成口服溶液、粉剂、颗粒剂、泡腾制剂、片剂、胶囊剂、缓释口服制剂；所述功能食品为酸奶、冰淇淋、果汁或米稀。
本发明应用丙烯酰胺灌胃的SD大鼠模拟丙烯酰胺暴露患者，来观察蓝莓花色苷抗丙烯酰胺诱导的海马和大脑皮层神经毒性的功效。研究发现，本发明提供的蓝莓花色苷可改善丙烯酰胺诱导的大鼠体重、摄食、饮水的减少及运动障碍；并通过抑制海马和皮层氧化应激和神经炎症，以减少神经元坏死和突触功能损伤。说明本发明提供的蓝莓花色苷能从多个方面改善丙烯酰胺神经毒性。本发明提供了一种安全、有效、易获取的改善丙烯酰胺神经毒性的治疗方案，也为蓝莓花色苷发掘了新的医疗用途。
附图说明
图1为实施例2中大鼠体重、摄食、饮水及运动能力；A为试验结束时代表性行为表现；B为试验第11、15、19天步态评分；C为体重变化；D为日均饲料摄入量；E为日均饮水量。
图2为实施例3中大鼠的海马和大脑皮层神经元组织形态学；A为苏木精-伊红(H&E)染色；B为坏死神经元百分比。
图3为实施例3中大鼠海马和大脑皮层突触体相关蛋白25(SNAP-25) 表达；A为Western blot检测条带；B为相对表达量。
图4为实施例4中大鼠海马、大脑皮层中丙二醛含量；A为海马中含量； B为大脑皮层中含量。
图5为实施例4中大鼠海马、大脑皮层中谷胱甘肽含量；A为海马中含量；B为大脑皮层中含量。
图6为实施例4中大鼠海马、大脑皮层中谷胱甘肽过氧化物酶含量；A 为海马中含量；B为大脑皮层中含量。
图7为实施例4中大鼠海马、大脑皮层中过氧化氢酶含量；A为海马中含量；B为大脑皮层中含量。
图8为实施例4中大鼠海马、大脑皮层中超氧化物歧化酶含量；A为海马中含量；B为大脑皮层中含量。
图9为实施例5中大鼠海马、大脑皮层小胶质细胞免疫组化染色；A为染色结果；B为阳性细胞相对数量百分比。
图10为实施例5中大鼠海马、大脑皮层星形胶质细胞免疫组化染色；A 为染色结果；B为阳性细胞相对数量百分比。
图11为实施例5中大鼠海马、大脑皮层炎症因子水平；A为白介素-1β (IL-1β)；B为白介素-6(IL-6)水平；C为肿瘤坏死因子-α(TNF-α)水平。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
将30g冷冻蓝莓果实在125mL甲醇、丙酮、水和甲酸混合物(体积比 40：40：20：0.1，甲醇、丙酮、水和甲酸均为纯物质)中均质(150bar,3×3 min)，获得总提取物。2000g离心10分钟，取上清液于40℃旋转蒸发。将蒸发后的沉淀溶解于20mL去离子水，用Sep-Pak活化的C18柱以两倍柱体积的酸化水(0.01％盐酸)洗涤以除去糖和酚酸，用两倍柱体积的乙酸乙酯(纯物质)洗脱其他酚类化合物，用两倍柱体积的酸化甲醇(0.01％盐酸) 洗脱花色苷。将含花色苷洗脱液低压干燥，得到蓝莓花色苷产品。
取1g制备的蓝莓花色苷溶解于3mL生理盐水中。用HPLC-MS检测蓝莓花色苷提取物可知，本实施例制备的蓝莓花色苷由矢车菊素-3-葡萄糖苷、矢车菊素-3，5-二葡萄糖苷和芍药色素-3-葡萄糖苷组成，三中花色苷组成比例约为90％、5％、5％。
实施例2
蓝莓花色苷改善丙烯酰胺诱导的大鼠体重、摄食、饮水及运动能力损失
材料与方法
使用前将实施例1中蓝莓花色苷溶解于生理盐水中，配置成1.75％的溶液。丙烯酰胺由美国Sigma Aldrich公司提供，称取7g丙烯酰胺溶于1L蒸馏水中，配置为0.7％溶液。8周龄SD雌性大鼠由北京维通利华公司提供。
将大鼠饲养于SPF屏障环境，保持恒温(22.0±1.0℃)、恒湿(55±5％)、 12小时昼夜交替以及自由摄食和饮水。适应性饲养1周后，随机将24只大鼠分成3组，即CON组、AA组及BAE+AA组，分别进行以下处理：①CON 组从实验第8天到第19天，以5mL/kg/天剂量灌胃生理盐水；②AA组从实验第8天到第19天，以35mg/kg/天剂量灌胃丙烯酰胺溶液；③BAE+AA 组从实验第1天到第19天，以175mg/kg/天剂量灌胃蓝莓花色苷溶液；并从第8天到第19天，以35mg/kg/天剂量灌胃丙烯酰胺溶液。实验期间，每天称量大鼠体重、摄食量和饮水量。实验结束后，大鼠用戊巴比妥钠过量麻醉处死。从实验开始后第11、15、19天进行步态评分。大鼠被放置在干净的空旷的场地上自由行动3min，由至少5名经过培训的实验无关人员对运动能力进行打分，其标准如下：1分为步态正常，没有受任何影响；2分为较轻微的步态异常，表现为在行走时有轻度足部外展和后肢无力；3分为中度步态异常，表现为行走时有明显的足部外展和后肢无力；4分为严重步态异常，表现为严重足部外展、不能正常行走、后肢拖曳，且无法支撑体重。所有数据以平均值±标准误表示，采用独立样本T检验比较所有组之间均值的差异。
结果
蓝莓花色苷可以缓解丙烯酰胺处理大鼠表现出的步态异常，包括足部张开、后肢扭转和行走困难(图1A)，并减少了中毒大鼠步态评分(图1B)。蓝莓花色苷还可以缓解丙烯酰胺处理大鼠的体重减轻(图1C)、摄食(图 1D)和饮水量减少(图1E)。表明蓝莓花色苷对丙烯酰胺诱导体重、摄食、饮水量减少及运动能力障碍有改善作用。
实施例3
蓝莓花色苷改善丙烯酰胺诱导的大鼠海马和皮层神经元及突触损伤。
材料与方法
SNAP-25、β-actin抗体由美国Santa Cruz公司提供。取实施例2大鼠大脑组织进行石蜡包埋、切片和H&E染色。在光学显微镜下观察，对每只大鼠海马DG、CA1、CA3和大脑皮层的坏死神经元和正常神经元进行计数，并计算损伤神经元数目。取实施例2大鼠大脑组织匀浆裂解并提取蛋白，用 Western blot方法检测大鼠海马和皮层突触蛋白SNAP-25，并以计算其相对表达量。所有数据以平均值±标准误表示，采用独立样本T检验比较所有组之间均值的差异。
结果
蓝莓花色苷改善了丙烯酰胺引发的神经元细胞核固缩、染色质深染、细胞排列松散无序等组织病理学损伤(图2A)，并减少了坏死神经元的数目 (图2B)。表明蓝莓花色苷可以抗丙烯酰胺诱导的海马和皮层神经元死亡。蓝莓花色苷改善了丙烯酰胺引发的突触蛋白SNAP-25的表达下调(图3)，提示蓝莓花色苷可以抗丙烯酰胺诱导的突触功能损伤。
实施例4
蓝莓花色苷改善丙烯酰胺诱导的大鼠海马、大脑皮层氧化应激。
材料与方法
ELISA试剂盒由上海酶联公司提供。取实施例3中大鼠大脑组织裂解液，用ELISA试剂盒检测海马、大脑皮层中丙二醛、谷胱甘肽、谷胱甘肽过氧化物酶、过氧化氢酶、超氧化物歧化酶的含量。所有数据以平均值±标准误表示，采用独立样本T检验比较所有组之间均值的差异。
结果
蓝莓花色苷减少了丙烯酰胺诱发的大鼠海马和皮层中脂质过氧化的生物标志物丙二醛的增加(图4)；还增加了丙烯酰胺诱发的大鼠海马和皮层中抗氧化物谷胱甘肽(图5)和抗氧化酶谷胱甘肽过氧化物酶(图6)、过氧化氢酶(图7)、超氧化物歧化酶(图8)含量的减少，表明蓝莓花色苷可抗丙烯酰胺诱发的氧化应激。
实施例5
蓝莓花色苷改善丙烯酰胺诱导的大鼠海马、大脑神经炎症。
材料与方法
离子钙结合适配器分子-1(IBA-1)和神经胶质纤维酸性蛋白(GFAP) 抗体由美国Santa Cruz公司提供，其分别为小胶质细胞和星形胶质细胞激活标志物；ELISA试剂盒由上海酶联公司提供。取实施例3中大鼠大脑组织切片，并分别进行IBA-1和GFAP免疫组化染色。在光学显微镜下观察，对每只大鼠海马DG、CA1、CA3和大脑皮层的阳性细胞统计。取实施例3中大鼠大脑组织裂解液，用ELISA试剂盒检测海马、大脑皮层中IL-1β、IL-6、 TNF-α含量。所有数据以平均值±标准误表示，采用独立样本T检验比较所有组之间均值的差异。
结果
蓝莓花色苷缓解了丙烯酰胺诱导的大鼠海马和大脑皮层中小胶质细胞 (图9)和星形胶质细胞(图10)的激活，并下调了炎症因子IL-1β、IL-6、 TNF-α的含量(图11)，表明蓝莓花色苷可抗丙烯酰胺诱发的神经炎症。
综上所述，蓝莓花色苷有效改善了丙烯酰胺诱导的体重、摄食、饮水减少及运动障碍；并通过抑制海马和皮层氧化应激和神经炎症，以减少神经元坏死和突触功能损伤，说明本发明能从多个方面改善丙烯酰胺神经毒性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种蓝莓花色苷在制备抗丙烯酰胺神经毒性的功能食品中的应用，其特征在于，所述神经毒性发生在海马和/或大脑皮层。
2.如权利要求1所述的应用，其特征在于，所述神经毒性包括海马和/或大脑皮层的神经元和突触损伤、氧化应激和神经炎症中的一种或几种。
3.一种权利要求1或2所述的蓝莓花色苷的制备方法，其特征在于，包括如下制备步骤：
(1)将蓝莓果实用提取剂提取，得到总提取物；
(2)所述总提取物经固液分离，得到上清液；
(3)浓缩所述上清液得到沉淀；
(4)所述沉淀加水配成溶液，经纯化、干燥后，得到蓝莓花色苷。
4.如权利要求3所述的制备方法，其特征在于，所述提取剂为甲醇、丙酮、水和甲酸按照体积比35～45：35～45：15～25：0.05～1.5配制的混合物；所述蓝莓果实与提取剂的质量体积比为25～35g：110～140mL。
5.如权利要求4所述的制备方法，其特征在于，所述提取为均质提取，所述均质提取的条件为100～200bar，2～4×2～4min。
6.如权利要求5所述的制备方法，其特征在于，所述纯化的方法为：用Sep-Pak C18柱分别以盐酸溶液、乙酸乙酯洗涤，然后用酸化甲醇洗脱；所述盐酸溶液的浓度为0.008～0.012％，用量为1.8～2.2倍柱体积；所述乙酸乙酯用量为1.8～2.2倍柱体积；所述酸化甲醇为含0.008～0.012％盐酸的甲醇溶液，用量为1.8～2.2倍柱体积。
7.一种权利要求1或2所述的蓝莓花色苷或按照权利要求3～6任一项所述的制备方法得到的蓝莓花色苷，其特征在于，所述蓝莓花色苷包括如下重量份的成分：矢车菊素-3-葡萄糖苷85～95份、矢车菊素-3，5-二葡萄糖苷1～10份、芍药色素-3-葡萄糖苷1～10份。
8.一种由权利要求7所述的蓝莓花色苷制备的功能食品，其特征在于，所述功能食品中所述蓝莓花色苷的含量为240～260mg/g。
9.如权利要求8所述的功能食品，特征在于，所述功能食品中还包括药学或食品生产可接受的载体或添加剂；所述药学或食品生产可接受的载体或添加剂包括：矫味剂、填充剂、润滑剂、粘合剂。
10.如权利要求8或9所述的功能食品，其特征在于，所述功能食品可制备成口服溶液、粉剂、颗粒剂、泡腾制剂、片剂、胶囊剂、缓释口服制剂；所述功能食品为酸奶、冰淇淋、果汁或米稀。
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| CN116076718A (zh) * | 2022-09-16 | 2023-05-09 | 上海青鋆健康科技有限公司 | 一种适合慢性肾衰竭人群使用的复方营养制剂及其制备方法和应用 |
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