CN115448989A - 一种老鹰茶多糖及其提取纯化方法和抗新冠肺炎病毒应用 - Google Patents
一种老鹰茶多糖及其提取纯化方法和抗新冠肺炎病毒应用 Download PDFInfo
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Abstract
本申请公开了医药技术领域中一种老鹰茶多糖，主要由甘露糖：葡萄糖醛酸：鼠李糖：葡萄糖：半乳糖：木糖：阿拉伯糖组成，其百分比为0.9％：0.6％：1％：22.6％：7.2％：4.3％：63.4％。研究表明，老鹰茶多糖具有抗新冠肺炎病毒的活性。
Description
技术领域
本发明涉及医药化学技术领域，尤其涉及一种老鹰茶多糖及其提取纯化方法和抗新冠肺炎病毒应用。
背景技术
本次新冠肺炎病毒SARS-Cov-2属于典型的冠状病毒，膜表面的刺突糖蛋白(S，Spike Protein)是与靶细胞受体结合的位点，Spike蛋白含有S1亚基和S2亚基。S1亚基主要包含有受体结合区(receptor binding domain，RBD)，S1亚基与靶细胞膜上的ACE2蛋白结合，具有结合细胞作用，是主要的抗原位点。针对新冠病毒的药物研发，包括病毒疫苗和抗体的研发，中药方剂的研发，化学药物的研发等。中药多糖是中药的活性成分之一，多糖具有多种寡糖末端，与糖蛋白结合域具有非常高的相似性，有报道称中药多糖具有很好的抗病毒活性，而且多糖具有多靶点，低毒副作用等生物活性，对于新型冠状病毒肺炎疾病(COVID-19)的预防和改善有很好的研究前景。
老鹰茶是由樟科，木姜子属，植物学名毛豹皮樟的叶或枝叶加工制作而成。在四川、重庆、云南、贵州、安徽等地十分普遍且饮用历史悠久，且无毒副作用，为药食同源植物。目前，对于老鹰茶多糖报道主要集中在抗氧化功能研究，因此，分类开发能充分发挥老鹰茶多糖功效是非常必要的。本专利以Spike/ACE2的配体受体结合通路为靶点，研究老鹰茶多糖的抗新冠病毒的生物功能及机制，为抗新冠肺炎病毒的药物研发提供新策略。
发明内容
本发明针对现有技术的不足，提供一种防治新冠肺炎的老鹰茶多糖。
本发明的目的之一是提供一种老鹰茶多糖，主要由甘露糖：葡萄糖醛酸：鼠李糖：葡萄糖：半乳糖：木糖：阿拉伯糖组成，其百分比为0.9％：0.6％：1％：22.6％：7.2％：4.3％：63.4％。
本发明的目的之二是提供一种老鹰茶多糖的提取纯化方法，具体为，称取老鹰茶，以水为溶剂加热提取，过滤，将滤液减压浓缩后离心，取上清液，将上清液透析后加4倍体积的无水乙醇，醇沉过夜，离心，加蒸馏水溶解沉淀，减压浓缩挥发乙醇，将浓缩液进行冷冻干燥，得到老鹰茶多糖。
进一步的，加热提取时，将老鹰茶放入煎药机中微沸煎煮提取4次，每次3.5h。
进一步的，加热提取时，先称取老鹰茶，第一次加入15倍量水，第二次加入10倍量水，第三次加入10倍量水，第四次加入10倍量水，每次煎煮后过滤备用。
进一步的，加热提取过滤所得滤液减压浓缩后的浓缩液，用玻璃纸透析48h。
本发明的目的之三是提供老鹰茶多糖在制备防治新冠肺炎药物中的应用。
本发明的工作原理及有益效果：以老鹰茶为原料，采用水提醇沉法，采用经透析纯化，冷冻干燥后得到老鹰茶多糖。得到的老鹰茶多糖能显著抑制A549细胞膜上的ACE2蛋白与 Spike-S1-FITC蛋白(FITC荧光蛋白结合的spike蛋白S1亚基基团)的结合，老鹰茶多糖可作为开发抗新冠肺炎新药的重要药物中间体，功能性保健食品的天然原料。
上述老鹰茶多糖在以Spike/ACE2的配体受体结合通路为靶点新型冠状病毒肺炎的生物功能及机制。
通过流式细胞仪检测A549细胞的平均荧光强度(X-Mean)，发现老鹰茶多糖能够抑制 Spike-S1-FITC蛋白与ACE2蛋白结合。
附图说明
图1为老鹰茶多糖单糖组成结果示意图；
图2为老鹰茶多糖抗新冠肺炎活性示意图。
具体实施方式
下面通过具体实施方式进一步详细说明：
1、老鹰茶多糖的制备
称取老鹰茶干燥叶片500g，第一次加入15倍量水，沸水煎煮3.5h，收集滤液，第二次加入10倍量水，沸水煎煮3.5h，第三次加入10倍量水，沸水煎煮3.5h，第四次加入10倍量水，沸水煎煮3.5h。合并四次滤液，80℃减压浓缩至1.5L，玻璃纸透析48h，透析后4000 rpm离心15min，取上清。缓慢加入4倍无水乙醇，边加边搅拌，室温放置过夜，4000rpm 离心15min得沉淀。用蒸馏水复溶沉淀，50℃减压浓缩将乙醇挥发，分装，冷冻干燥，即得老鹰茶多糖21g。
2、老鹰茶多糖HTP的单糖组成分析
HPLC条件：流动相组成为乙腈：磷酸缓冲盐＝18.2:81.8。检测器型号：依利特EClassical 3200，检测波长：254nm。色谱柱型号：Supersil ODS2(4.6×250mm)；柱温：35℃。进样量：20μL，流速：1mL/min。
样品前处理：
(1)酸水解：称取2mg老鹰茶多糖样品置于棕色小瓶中，使其溶于2mL 4M的三氟乙酸(TFA)，加盖密封。在110℃的条件下加热水解4h，取出冷却至室温，使用甲醇减压浓缩，重复操作直至除尽TFA。蒸干后，残渣溶解200μL蒸馏水中。
(2)PMP衍生化：取100μL(1)中的完全酸水解溶液，加入浓度为0.6M的NaOH 溶液100μL混匀，再加入200μL现配置的浓度为0.5M的PMP甲醇溶液，密封后涡漩混匀。水浴温度为70℃条件下反应100min后，冷却至室温，用浓度为0.3M的HCl 200μL进行中和，加蒸馏水补足至1mL。再加入1mL三氯甲烷萃取，取出上层水相，多次重复萃取，尽量除尽PMP。涡旋后取20μL上清液进样分析。
(3)取100μL的1mg/mL单糖标准品(Man，Rha，Gal，GalA，Glc，GlcA，Xyl， Ara，Fuc)混合液，按样品PMP衍生同法处理，然后进行HPLC分析。
如图1所示，与混标进行对比，老鹰茶多糖主要由甘露糖：葡萄糖醛酸：鼠李糖：葡萄糖：半乳糖：木糖：阿拉伯糖组成，其百分比为0.9％：0.6％：1％：22.6％：7.2％：4.3％：63.4％。
3、老鹰茶多糖抗新冠肺炎病毒活性测定
人肺癌A549细胞培养于DMEM完全培养基，于37℃、5％CO2饱和湿度的孵箱中贴壁培养，隔天换液，显微镜下观察待融合度达到90％后，用0.25％胰蛋白酶消化传代。
人肺癌A549细胞传代3代后，将处于对数生长期的细胞用胰蛋白酶消化，用70％乙醇于4℃固定12h。将固定后的细胞用PBS制备为细胞悬液后计数。
通过流式细胞仪检测老鹰茶多糖对Spike-S1-FITC蛋白与ACE2蛋白结合的抑制作用。
取一定量细胞悬液至EP管，设为空白组。取等量细胞悬液至EP管，加入Spike-S1-FITC 蛋白，设为模型组。取等量细胞悬液至EP管，加入Spike-S1-FITC蛋白，加入老鹰茶多糖溶液，使药物浓度为0.5mg/mL，设为实验组。
将离心管置于4℃摇床，结合12h后，离心，留沉淀。用PBS重悬，混匀。通过流式细胞仪检测细胞的平均荧光强度(X-Mean)，以判断模型组与实验组细胞与Spike-S1-FITC 蛋白的结合情况。
如图2所示，模型组的A549细胞平均荧光强度(X-Mean)为20.2，显著高于阴性对照组。与模型组相比，实验组(老鹰茶多糖处理组)光密度显著降低，可知老鹰茶多糖能够显著抑制Spike-S1-FITC蛋白与A549细胞(肺细胞系)结合，老鹰茶多糖可以被开发成抗新冠肺炎病毒的药物。
以上所述的仅是本发明的实施例，方案中公知的具体结构及特性等常识在此未作过多描述。应当指出，对于本领域的技术人员来说，在不脱离本发明结构的前提下，还可以作出若干变形和改进，这些也应该视为本发明的保护范围，这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准，说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (6)
1.一种老鹰茶多糖，其特征在于，主要由甘露糖：葡萄糖醛酸：鼠李糖：葡萄糖：半乳糖：木糖：阿拉伯糖组成，其百分比分别为0.9％：0.6％：1％：22.6％：7.2％：4.3％：63.4％。
2.根据权利要求1所述的一种老鹰茶多糖的提取纯化方法，其特征在于，称取老鹰茶，以水为溶剂加热提取，过滤，将滤液减压浓缩后离心，取上清液，将上清液透析后加4倍体积的无水乙醇，醇沉过夜，离心，加蒸馏水溶解沉淀，减压浓缩挥发乙醇，将浓缩液进行冷冻干燥，得到老鹰茶多糖。
3.根据权利要求2所述的一种老鹰茶多糖的提取纯化方法，其特征在于，加热提取时，将老鹰茶放入煎药机中微沸煎煮提取4次，每次3.5h。
4.根据权利要求3所述的一种老鹰茶多糖的提取纯化方法，其特征在于，加热提取时，先称取老鹰茶，第一次加入15倍量水，第二次加入10倍量水，第三次加入10倍量水，第四次加入10倍量水，每次煎煮后过滤备用。
5.根据权利要求4所述的一种老鹰茶多糖的提取纯化方法，其特征在于，加热提取过滤所得滤液减压浓缩后的浓缩液，用玻璃纸透析48h。
6.根据权利要求1所述的老鹰茶多糖在制备防治新冠肺炎药物中的应用。
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