CN111529711A - 一种抗血小板聚集的芦荟提取物及其在prp制备中的应用 - Google Patents
一种抗血小板聚集的芦荟提取物及其在prp制备中的应用 Download PDFInfo
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Abstract
一种抗血小板聚集的芦荟提取物及其在PRP制备中的应用，包括以下步骤：芦荟提取物的制备、抗ADP诱导的血小板聚集的检测、PRP的制备与激活。本发明的芦荟提取物对ADP诱导的血小板聚集有显著的抑制作用，与枸橼酸钠合用抗凝剂制备得到PRP浓度更高，活性更强，激活后PRP可用于伤口修复等相关临床应用。
Description
技术领域
本发明属于生物医药技术领域，涉及一种抗血小板聚集的芦荟多糖及其在 PRP制备中的应用。
背景技术
富血小板血浆(platelet rich plasma，PRP)来自于自体全血或者脐血，其相对于全血/脐血含有更高倍数浓度的血小板，因制备简便快速、风险较小及生物功能显著等优点，被广泛用于组织修复再生、伤口感染治疗和机体功能重建等方面。PRP通过释放大量机体所需的生长因子和细胞因子，同时参与了血小板凝固和纤维蛋白产生的止血和免疫应答进程。因此PRP发挥临床治疗作用主要是由于其分泌的蛋白分子和细胞因子等成分。而目前尚无统一标准来规范PRP的制备，不同制备技术获得的PRP所含的血小板浓度和组分也不同。
常用的PRP制备方法是二次离心法，是将血液注入含抗凝剂的试管内，第一次离心分为3层，最低层为红细胞，中间层为含富血小板的黄衣层，最上层为贫血小板血浆，用移液器吸取最上层血浆及中间层后注入另一个不含抗凝胶的试管中，再次离心重新分为三层，最低层为少量红细胞，弃用3/4上层血浆后用剩余血浆复溶中间黄衣层得到富血小板血浆。
最常用的抗凝剂有肝素钠、枸橼酸钠、EDTA盐、草酸盐等，然而这些抗凝剂或多或少具有明显的缺点，如钠离子存在易导致血浆电解质混乱、肝素诱导血小板减少症及骨质疏松症、枸橼酸钠导致Ca2+离子失衡等，特别用于大量人体输入时产生的副作用更加明显，因此研发筛选新的天然抗凝剂具有重要的实际价值。
芦荟提取物有开发成新型抗凝剂的潜力，芦荟具有抗衰老、促进愈合、强心活血、消炎杀菌等多种药理作用，其成分复杂，含有多糖、黄酮、蒽醌类、异柠檬酸钙多种生物活性物质，在制备PRP过程中，血液运输、血小板富集等过程所需时间较长，很难保证血小板不被激活，血液不被细菌污染，因此具有多种生物活性的芦荟提取物是比较符合PRP制备过程中抗凝剂选择要求。
本研究研究了芦荟提取物抗血小板聚集、凝血的作用效果，评价了其作为抗凝剂制备得到的PRP中血小板的含量和活性，有望开发出用于伤口愈合效果更好的PRP制剂。
发明内容
为了克服现有技术的不足，本发明提供了一种抗血小板聚集的芦荟提取物及其在PRP制备中的应用，芦荟提取物具有显著的抗血小板凝集作用，作为抗凝剂制备得到PRP制剂与枸橼酸钠相比，其细胞因子含量及活性更高，具有更好的治疗效果。
为实现上述目的，本发明提供如下技术方案：一种抗血小板聚集的芦荟提取物及其在PRP制备中的应用，包括以下步骤：其中，芦荟提取物的制备方法包括以下步骤：取芦荟粉末，按10:1体积加入水，100℃回流提取，过滤后，浓缩冻干得芦荟提取物。
本发明所提供的芦荟提取物，经测试具有明显抑制凝血酶、ADP、胶原、花生四烯酸诱导的血小板聚集的作用，且呈现剂量依赖性。
所述的抗血小板聚集的芦荟提取物可应用于制备抗血小板聚集及治疗心血管疾病药物。
所述的抗血小板聚集的芦荟提取物可应用于制备抗血小板聚集及治疗心血管疾病药物中，该芦荟提取物为活性组分，可制备成其他制剂如注射剂、片剂、丸剂等。
所述的抗血小板聚集的芦荟提取物可应用于制备PRP中，与枸缘酸钠联用作为抗凝剂复合物可降低制备过程中血小板的过早激活及防止细菌污染。
所述的抗血小板聚集的芦荟提取物与枸缘酸钠联用作为抗凝剂复合物制备的得到PRP后，经CaCl2和凝血酶激活后，可应用于止血、抗炎、促进伤口愈合、改善血供等领域。
附图说明
为了使本发明的内容更容易被清楚地理解，下面根据具体实施例并结合附图，对本发明作进一步详细的说明。
图1为芦荟提取物对凝血酶、ADP、胶原、花生四烯酸诱导的血小板聚集的影响效果条状图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：芦荟提取物的制备
称取芦荟粉末100g，加入圆底烧瓶中，再加入1L双蒸水，搅拌混匀，置于水浴锅中加热回流，100℃加热3h，先棉纱过滤，再3000rpm离心 3min，取上清液，减压旋蒸至100mL，至于-80℃冰箱过夜，再冻干48h，得芦荟提取物。
实验例2：通过光学比浊法检测芦荟提取物对凝血酶、ADP、胶原、花生四烯酸诱导的抗血小板聚集的影响。
采用常用二次离心法从大鼠血液制备得到富血小板血浆，取100μL 富血小板血浆，采用PBS缓冲溶液(pH＝7.4)血小板数目调整为2.5×108个/mL。取400μL血小板重悬液，于37℃下预热5min后，加入芦荟提取物(终浓度为0g/mL，0.01g/L，0.1g/L，1g/L)37℃孵育20min。将含有芦荟提取物的血小板血浆置于血小板聚集仪测试区，调零后，加入凝血酶、ADP、胶原、花生四烯酸刺激剂，在1200rpm条件下搅拌10min，用生理盐水作对照，测定血小板聚集率，结果发现芦荟提取物能明显降低凝血酶、ADP、胶原、花生四烯酸对血小板诱导聚集效果(图1)。
实例3：芦荟提取物与枸缘酸钠联用作为抗凝剂复合物
抗凝剂复合物的制备是枸缘酸钠与芦荟提取物的质量比例为1：1，再配制枸缘酸钠浓度为4％的溶液。将抗凝剂溶液滴加入抗凝管中，震摇挂壁后干燥，按1mL血液滴加20μL使用。把受试者的全血采集到含有抗凝剂的试管中，充分混匀后离心，取100μL以计算血小板数量，分配到离心管后进行离心，200g离心4min，分三层后，利用移液器把最上层和大部分中间层抽出到新的离心管中，再次离心，转速调为1500g，离心 20min，吸取3/4的上层血浆，剩余1/4血浆重悬沉淀下来的血小板，并用吸取3/4的上层血浆定溶在4mL刻度，获得富血小板血浆(PRP)，抽取100μL进行血小板计数。经计算分离前全血中血小板浓度为1.8×108/mL，全血体积为5mL，总血小板数量为9×108。只采用枸缘酸钠作为抗凝剂，分离得到血小板数量为7.3×108，回收率为81.11％。相反，采用枸缘酸钠和芦荟提取物作为联用抗凝剂，同样步骤和方法，分离得到的血小板数量为8.2×108，回收率为91.11％。说明采用芦荟提取物可以显著增加PRP中的含量和回收率。
实列4：芦荟提取物对血小板活性的影响。
针对实施3制备的两种PRP，再次离心2000g，除去上层血浆，加入 PBS缓存液配制成浓度为1×108个/mL，分别取0.5mL血小板溶液，按 10：1体积加入激活剂，激活剂含2.5g/L的CaCl2和1000U/mL牛凝血酶。迅速混匀后，37℃放置1h后于4℃冰箱过夜，试管离心，5000rpm离心 30min，取上清液，测定上清液中的三种细胞因子，即FGF(成纤维细胞生长因子)、TGF-β1(转化生长因子-β1)、PDGF(血小板衍生生长因子)的量 (pg/ml)，结果见表1。
表1芦荟提取物对血小板活性的影响
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
Claims (4)
1.一种抗血小板聚集的芦荟提取物，其特征在于：取芦荟粉末，通过水提法进行提取得到提取液，过滤除去沉淀，浓缩冻干得到芦荟提取物。
2.按权利要求1所述的一种抗血小板聚集的芦荟提取物在制备抗ADP诱导的血小板聚集PRP制剂中的应用。
3.按权利要求2所述的应用，其特征在于所述芦荟提取物单独或与肝素钠、枸橼酸钠联用作为抗凝剂，制备PRP制剂。
4.按权利要求3所述的制剂，可应用于止血、抗炎、促进伤口愈合、改善血供等领域。
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