CN113861449B - 一种茯苓多糖水凝胶及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种茯苓多糖水凝胶及其制备方法和应用,其制备方法包括如下步骤:将碱溶性茯苓多糖溶于碱液中,加酸调至pH<11,制备浓度≥4mg/mL的碱溶性茯苓多糖分散液;将所得碱溶性茯苓多糖分散液于不高于50℃的条件下静置,得茯苓多糖水凝胶。本发明的茯苓多糖水凝胶的制备方法简单、生产成本低,所制备的茯苓多糖水凝胶具有良好的力学性能、保水性和抗炎性,该茯苓多糖水凝胶负载的药物可在50小时左右释放出70%~80%,因此,该茯苓多糖水凝胶作为药物控缓释放载体具有良好的应用前景。
Description
技术领域
本发明涉及生物高分子材料领域,特别涉及一种茯苓多糖水凝胶及其制备方法和应用。
背景技术
凝胶是一种通过共价键或非共价键交联形成的具有三维网络结构的软固体。近年来,凝胶由于具有良好的保水性、生物相容性、可降解性和环境响应性,已被广泛应用于药物释放系统,尤其是对于一些需要持续治疗的慢性疾病具有较大潜力。然而,目前大多数的凝胶基质都为合成材料,更重要的是与所载药物几乎无协同作用。
多糖是由多单糖通过糖苷键连接而成的一类天然高分子化合物,广泛存在于动物细胞膜、植物和微生物细胞壁中。它不仅具有调节免疫作用,还在抗肿瘤、抗氧化、降血脂等方面发活着广泛的药理作用。多糖分子间因存在丰富的结合位点和良好的生物相容性,因此是制备天然水凝胶的优良材料。将其用作药物释放系统,不仅可减小无效载体的使用,还可有望实现“药辅协同”的作用。
茯苓Poria cocos(Schw.)Wolf为多孔菌科真菌茯苓的烘干菌核,是我国的传统中药,也是药食两用的大宗中药材。茯苓多糖是茯苓药材的主要组成部分,其含量占茯苓菌核的70%~90%。现代药理学研究表明,茯苓多糖是茯苓的主要活性成分,具有抗肿瘤、提高免疫等功效。目前,有关茯苓多糖的研究主要集中在药理活性和作用机理方面,还未见利用茯苓多糖制备凝胶的相关研究。
发明内容
发明人发现,一定浓度的碱溶性茯苓多糖分散液在pH<11、温度小于50℃的条件下静置即可得到茯苓多糖水凝胶。该茯苓多糖水凝胶在<50℃条件下热稳定,其浸出液具有生物活性,且在37℃下、pH=5.8~7.4的条件下可在50小时左右释放出70%~80%的负载药物,据此,可将该茯苓多糖水凝胶用于制备缓释药物。
本发明提供的技术方案具体如下:
第一方面,一种茯苓多糖水凝胶的制备方法,包括如下步骤:
将碱溶性茯苓多糖溶于碱液中,加酸调至pH<11,制备浓度≥4mg/mL的碱溶性茯苓多糖分散液;将所得碱溶性茯苓多糖分散液在温度小于50℃的条件下静置,得茯苓多糖水凝胶。
碱液的温度优选为优选为≤35℃,进一步优选为≤30℃,更进一步优选为≤25℃;碱溶性茯苓多糖分散液静置时的温度优选为≤35℃,进一步优选为≤30℃,更进一步优选为≤25℃,最优选为4℃。
作为上述技术方案的优选,碱溶性茯苓多糖分散液的浓度为4~30mg/mL。
作为上述技术方案的优选,碱溶性茯苓多糖分散液的浓度为C1时,控制0<pH≤1;碱溶性茯苓多糖分散液的浓度为C2时,控制0<pH≤7.8;碱溶性茯苓多糖分散液的浓度为C3时,控制0<pH≤10;其中,4mg/mL≤C1<10mg/mL,10mg/mL≤C2<25mg/mL,C3≥25mg/mL。
作为上述技术方案的优选,碱溶性茯苓多糖分散液4≤pH≤6,该pH条件下碱溶性茯苓多糖形成茯苓多糖水凝胶的时间<30s。
作为上述技术方案的优选,碱溶性茯苓多糖分散液的浓度为1wt%±0.1wt%、pH=7±0.2,该条件下制备的茯苓多糖水凝胶对药物的释放时间长达50小时,且制备的茯苓多糖水凝胶pH比较温和,完全凝胶化的时间<45min。
作为上述技术方案的优选,碱液为氢氧化钠溶液或氢氧化钾溶液,所述酸为盐酸、磷酸、硫酸、柠檬酸、冰乙酸中的至少一种。
作为上述技术方案的优选,碱溶性茯苓多糖通过如下方法制备得到:
茯苓干粉用乙醇浸提,烘干滤渣,得一次茯苓渣;
一次茯苓渣水煮,烘干滤渣,得二次茯苓渣;
二次茯苓渣用碱液浸提,过滤除去滤渣,所得滤液加酸调pH、静置,得沉淀物;
沉淀物用纯水脱盐后烘干,得碱溶性茯苓多糖。
第二方面,本发明提供一种茯苓多糖水凝胶,该茯苓多糖水凝胶通过上述茯苓多糖水凝胶的制备方法制备得到,该茯苓多糖水凝胶弹性模量大、强度大,能够释放出具有生物活性的茯苓多糖。
第三方面,本发明提供上述茯苓多糖水凝胶在制备缓释药物中的用途,具体地,在碱液中分散药物,且该药物在碱液pH变化时不改变分散状态和活性;茯苓多糖水凝胶制备的缓释药物在pH5.8~7.4条件下、可在50小时左右将药物释放70%~80%,且该茯苓多糖水凝胶释放出具有生物活性的茯苓多糖。
与现有技术相比,本发明具有如下优点和有益效果:
(1)本发明首次提出以碱溶性茯苓多糖为原料制备具有三维网络结构的茯苓多糖水凝胶,该茯苓多糖水凝胶具有良好力学性能、保水性和抗炎性。
(2)本发明利用茯苓多糖水凝胶制备的缓释药物可在50小时左右释放出70%~80%的药物,且该茯苓多糖水凝胶释放出具有生物活性的茯苓多糖。
(3)本发明提供的茯苓多糖水凝胶的制备方法绿色环保、工艺简单、成本低,有望运用在药物控缓释领域,拓展了茯苓多糖的应用范围。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1中碱溶性茯苓多糖分散液和茯苓多糖水凝胶的宏观照片;图1(a)为碱溶性茯苓多糖分散液的宏观照片,图1(b)为茯苓多糖水凝胶的宏观照片。
图2为本发明实施例1中茯苓多糖水凝胶的扫描电镜图。
图3展示了不同pH的碱溶性茯苓多糖分散液的最低凝胶化浓度。
图4展示了不同浓度碱溶性茯苓多糖分散液制备的茯苓多糖水凝胶的相转变温度。
图5显示了不同pH、浓度为2.5wt%的碱溶性茯苓多糖分散液的凝胶化时间。
图6为茯苓多糖水凝胶的储能模量和损耗模量的曲线图。
图7展示了茯苓多糖水凝胶浸出液按1:1、1:2、1:10、1:20倍稀释后培养24h的RAW264.7细胞的细胞活力。
图8为茯苓多糖水凝胶对LPS诱导RAW264.7细胞生成肿瘤坏死因子(TNF-α)炎症因子水平的影响。
图9为茯苓多糖水凝胶对LPS诱导RAW264.7细胞生成白细胞介素1β(IL-1β)炎症因子水平的影响。
图10为茯苓多糖载药凝胶在37℃、不同pH条件下对黄芩苷的释放曲线。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合具体实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明的一部分实施例,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的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。
本发明从茯苓药材中提取碱溶性茯苓多糖,将一定量的碱溶性茯苓多糖溶于碱液中,制备碱溶性茯苓多糖分散液;向碱溶性茯苓多糖分散液中加酸调至pH<11,于室温或4℃下静置,得茯苓多糖水凝胶。
以上技术方案中的碱液为氢氧化钠溶液或氢氧化钾溶液等强碱性溶液,碳酸盐溶液无法使碱溶性茯苓多糖溶解。
以上技术方案中的酸采用磷酸、盐酸、硫酸、柠檬酸或醋酸,为避免加酸后碱溶性茯苓多糖分散液被过度稀释,建议采用浓磷酸、浓盐酸、浓硫酸或冰醋酸,以下实施例中均采用浓磷酸。
以上技术方案中,加酸调节pH之后碱溶性茯苓多糖分散液需保持浓度在一定范围内,浓度太低时,碱溶性茯苓多糖分散液一直保持溶胶状态,无法生成水凝胶;浓度太高时,碱溶性茯苓多糖分散液难以完全溶解;且碱溶性茯苓多糖分散液需保持静置,否则容易出现网络结构破坏,而无法形成交联的水凝胶。
具体地,将碱溶性茯苓多糖复溶于0.3~2mol/L氢氧化钠溶液中,加浓酸调节pH值至1~9,并保证调节pH值后碱溶性茯苓多糖的浓度为2wt%~3wt%,于室温或4℃下静置5s~3h,即得到茯苓多糖水凝胶。
本发明从茯苓药材中提取碱溶性茯苓多糖的步骤如下:
(1)将茯苓药材粉碎,过40目筛,用100%乙醇浸泡12h后烘干滤渣,得一次茯苓渣;一次茯苓渣用20倍水煮2h后过滤弃掉滤液,重复3次,烘干滤渣,得二次茯苓渣;
(2)按照1:30料液重量比将二次茯苓渣分散在0.5~2mol/L氢氧化钠溶液中,室温搅拌,过滤除去滤渣,取滤液加1mol/L盐酸调节pH至7,静置1h后,将沉淀物用纯水脱盐、干燥,得到碱溶性茯苓多糖。
实施例1:茯苓多糖水凝胶的制备
(1)将茯苓药材打粉,过40目筛;先乙醇浸提:用100%乙醇浸泡12h后过滤,烘干滤渣,得一次茯苓渣;再热水浸提:一次茯苓渣用20倍水煮2h后过滤,弃掉滤液;重复热水浸提步骤3次,将滤渣烘干,得到二次茯苓渣;
(2)按照1:30料液重量比将二次茯苓渣分散于0.5mol/L氢氧化钠溶液中,室温搅拌,过滤除去滤渣;向滤液中加1mol/L盐酸调节pH至7,静置1h后,滤出沉淀物;将沉淀物用纯水脱盐、烘干,得到碱溶性茯苓多糖;
(3)将碱溶性茯苓多糖复溶于0.5mol/L氢氧化钠溶液中,然后加浓磷酸调节pH至1,得浓度为2wt%的碱溶性茯苓多糖分散液,碱溶性茯苓多糖分散液于室温下静置0.5h,得到茯苓多糖水凝胶。
用数码相机记录碱溶性茯苓多糖分散液和茯苓多糖水凝胶的外观,结果如图1所示,图1(a)为碱溶性茯苓多糖分散液的宏观照片,图1(b)为茯苓多糖水凝胶的宏观照片。将该茯苓多糖水凝胶冷冻干燥后,粘到导电胶上,喷金后进行电镜扫描,图2为本实施例中茯苓多糖水凝胶的电镜照片,如图2所示,茯苓多糖水凝胶具有典型的三维网络结构。
实施例2:凝胶化条件选择
将实施例1得到的碱溶性茯苓多糖复溶于0.5mol/L氢氧化钠溶液中,加浓磷酸调节pH,制备不同浓度的碱溶性茯苓多糖分散液。讨论不同凝胶化pH下,碱溶性茯苓多糖分散液的最低凝胶化浓度。
实验结果表明,碱溶性茯苓多糖分散液的凝胶化行为具有pH依赖性:当碱溶性茯苓多糖分散液≤0时,难以凝胶;当碱溶性茯苓多糖分散液0<pH≤1时,其最低凝胶化浓度为4mg/mL,且pH越接近0最低凝胶化浓度越高,接近16mg/mL;当碱溶性茯苓多糖分散液1.5<pH<7.8时,其最低凝胶化浓度为9mg/mL;当碱溶性茯苓多糖分散液pH=7.8时,其最低凝胶化浓度为10mg/mL;当碱溶性茯苓多糖分散液pH=10.50时,其最低凝胶化浓度为25mg/mL;当碱溶性茯苓多糖分散液调至pH=11.3左右时,碱溶性茯苓多糖分散液处于半凝胶的状态,无法完全倒置不流动。图3展示了不同pH下碱溶性茯苓多糖分散液的最低凝胶化浓度。
实施例3
将实施例1得到的碱溶性茯苓多糖复溶于0.5mol/L氢氧化钠溶液中,加浓磷酸调节pH=1,制备不同浓度(4mg/mL、8mg/mL、12mg/mL、16mg/mL、20mg/mL)的碱溶性茯苓多糖分散液,碱溶性茯苓多糖分散液于室温下静置0.5h,得到茯苓多糖水凝胶。加热茯苓多糖水凝胶,探讨不同浓度的碱溶性茯苓多糖分散液制备的茯苓多糖水凝胶的瓦解温度(即相转变温度TGS)。
图4所示的相转变温度实验结果显示,茯苓多糖水凝胶的相转变温度与碱溶性茯苓多糖分散液的浓度呈正相关。当碱溶性茯苓多糖分散液浓度>8mg/mL时,所得茯苓多糖水凝胶的相转变温度TGS为52℃;当碱溶性茯苓多糖分散液浓度>12mg/mL时,所得茯苓多糖水凝胶的相转变温度TGS为58℃;当碱溶性茯苓多糖分散液浓度>16mg/mL时,所得茯苓多糖水凝胶的相转变温度TGS为62℃;当碱溶性茯苓多糖分散液浓度>20mg/mL时,所得茯苓多糖水凝胶的相转变温度TGS为64℃。
实施例4
将实施例1得到的碱溶性茯苓多糖复溶于0.5mol/L氢氧化钠溶液中,加浓磷酸调节pH为0~10,制备浓度为25mg/mL的碱溶性茯苓多糖分散液,碱溶性茯苓多糖分散液于室温下静置,得到茯苓多糖水凝胶,记录不同pH的碱溶性茯苓多糖分散液从静置到倒置完全倒置不流动时的时间,即为碱溶性茯苓多糖分散液的凝胶化时间。
表1展示了不同pH的25mg/mL碱溶性茯苓多糖分散液形成茯苓多糖水凝胶所需的时间,结果如图5所示。
表1
pH(25mg/mL) | 所需时间 |
0.62 | 30min |
0.95 | 12min |
2.14 | 20-30s |
3.19 | 20s |
4.2 | 4s |
5.25 | 5s |
6.06 | 5s |
7 | 15~20min |
7.79 | 30min |
10.17 | 40min |
10.5 | 3h |
实施例5:茯苓多糖水凝胶的流变性能实验
将实施例1得到的碱溶性茯苓多糖复溶于0.5mol/L氢氧化钠溶液中,加浓磷酸调节pH至1,制备不同浓度(1wt%、1.5wt%、2.0wt%、2.5wt%)的碱溶性茯苓多糖分散液;将碱溶性茯苓多糖分散液于室温下静置0.5h,得到茯苓多糖水凝胶。采用流变仪在25℃下测定,选取25mm的夹具,设定振幅为1%、两板间距为1mm。在频率扫描的测定模式下,以角频率为0.1~10rad/s测定茯苓多糖水凝胶的储能模量G'和损耗模量G"。
图6为本实施例中茯苓多糖水凝胶的流变学性能,如图6所示,在角频率为0.01~10rad/s时,制备得到的茯苓多糖水凝胶的储能模量G'大于损耗模量G",由此证明该条件下形成了茯苓多糖水凝胶,且具有良好的弹性性能。随着碱溶性茯苓多糖分散液浓度的增加,G'和G"都随之增加,表明茯苓多糖水凝胶的力学性能越好。
实施例6:茯苓多糖水凝胶的抗炎性实验
将实施例1得到的碱溶性茯苓多糖复溶于0.5mol/L氢氧化钠溶液中,加浓磷酸调节pH至7,制备浓度为2wt%的碱溶性茯苓多糖分散液;将碱溶性茯苓多糖分散液于室温下静置2h,得到茯苓多糖水凝胶。
采用巨噬细胞RAW264.7作为研究对象,利用脂多糖(LPS)进行刺激诱导炎症发生以造成细胞炎症反应模型。RAW264.7细胞培养在37℃、5%CO2饱和湿度环境下,于胎牛血清的DMEM培养基中培养。在0.25g茯苓多糖水凝胶上方加入胎牛血清的DMEM培养基,浸泡24h,得到茯苓多糖水凝胶的浸提液;将细胞培养在茯苓多糖水凝胶的浸提液中,以1μg/mL LPS刺激24h后,观察茯苓多糖水凝胶的浸提液对细胞炎症的反应。
表2抗炎性实验分组
提取细胞内miRNA,加入Trizol充分裂解后,按200μL氯仿/mL Trizol加入氯仿充分混合,12000rpm离心15min,取滤液加入80%体积的异丙醇,混匀后12000rpm离心10min。将沉淀溶解于无miRNA酶的75%乙醇中。测定RNA浓度后,按照试剂盒说明书,取1.0μg RNA进行逆转录,以荧光实时定量PCR仪检测TNF-α和IL-1β的miRNA表达。各基因上下游引物序列分别为,TNF-α:5'-GGCGGTGCCTATGTCTCA-3'和5'-CCTCCACTTGGTGGTTTGT-3';IL-1β:5'-GTTCCCATTAGACAACTGC-3'和5'-GATTCTTTCCTTTGAGGC-3';GAPDH:5'-TGTTTCCTCGTCCCGTAGA-3'和5'-GATGGCAACAATCTCCACTTTG-3'。以GAPDH作为内参基因,将目的基因饱和状态循环数值与GAPDH基因饱和状态循环数值的差作为基础,计算目的基因表达差异。
图7展示了茯苓多糖水凝胶的浸提液分别按1:1、1:2、1:10、1:20倍稀释后得到的培养基培养24h的RAW 264.7细胞的细胞活力。
图8为茯苓多糖水凝胶对LPS诱导RAW264.7细胞产生肿瘤坏死因子-α(TNF-α)水平的影响,从图8中可以看出:与空白组比,LPS组TNF-α水平显著增加(p<0.001),说明LPS刺激RAW264.7细胞产生TNF-α水平明显增加;与LPS组相比,茯苓多糖水凝胶组TNF-α水平降低(p<0.05),这说明茯苓多糖水凝胶组能抑制TNF-α的表达水平。
图9为茯苓多糖水凝胶对LPS诱导RAW264.7细胞产生白细胞介素1β(IL-1β)水平的影响,从图9中可以看出:与空白组比,LPS组IL-1β水平显著增加(p<0.001),说明LPS刺激RAW264.7细胞产生IL-1β水平明显增加;与LPS组相比,茯苓多糖水凝胶组IL-1β水平明显降低(p<0.001),这说明茯苓多糖水凝胶组能显著抑制IL-1β的表达水平。
分析认为,茯苓多糖水凝胶中包裹有一定量的茯苓多糖水溶胶,在胎牛血清的DMEM培养基的浸泡下,茯苓多糖水溶胶缓慢渗出。
实施例7:茯苓多糖水凝胶在体外释药中的应用
将实施例1得到的碱溶性茯苓多糖复溶于含有1.5mg/mL黄芩苷的0.5mol/L氢氧化钠溶液中,加浓磷酸调节pH至7,制备浓度为1wt%的碱溶性茯苓多糖分散液;将碱溶性茯苓多糖分散液于室温下静置2h,即得到茯苓多糖载药凝胶。
图10展示了茯苓多糖载药凝胶在37℃下、不同释放介质环境下的药物释放行为。从图10中可以看出,茯苓多糖载药凝胶可以有效实现黄芩苷的长程释放和控释,说明本发明制备的茯苓多糖水凝胶可用作药物的载体。
以上所述仅是本发明的具体实施方式,使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所申请的原理和新颖特点相一致的最宽的范围。
Claims (9)
1.一种茯苓多糖水凝胶的制备方法,其特征在于,包括如下步骤:
将碱溶性茯苓多糖溶于碱液中,加酸调至pH<11,制备浓度≥4mg/mL的碱溶性茯苓多糖分散液;将所得碱溶性茯苓多糖分散液于不高于50℃的条件下静置,得茯苓多糖水凝胶;
碱溶性茯苓多糖分散液的浓度为C1时,控制0<pH≤1;碱溶性茯苓多糖分散液的浓度为C2时,控制0<pH≤7.8;碱溶性茯苓多糖分散液的浓度为C3时,控制0<pH≤10;其中,4mg/mL≤C1<10mg/mL,10mg/mL≤C2<25mg/mL,C3≥25mg/mL。
2.根据权利要求1所述的茯苓多糖水凝胶的制备方法,其特征在于:所述碱溶性茯苓多糖分散液4≤pH≤6。
3.根据权利要求1所述的茯苓多糖水凝胶的制备方法,其特征在于:所述碱溶性茯苓多糖分散液的浓度为4~30mg/mL。
4.根据权利要求1所述的茯苓多糖水凝胶的制备方法,其特征在于:所述碱溶性茯苓多糖分散液静置时的温度≤30℃。
5.根据权利要求1所述的茯苓多糖水凝胶的制备方法,其特征在于:所述碱液为氢氧化钠溶液或氢氧化钾溶液,所述酸为盐酸、磷酸、硫酸、柠檬酸、冰乙酸中的至少一种。
6.根据权利要求1所述的茯苓多糖水凝胶的制备方法,其特征在于:所述碱溶性茯苓多糖通过如下方法制备得到:
茯苓干粉用乙醇浸提,烘干滤渣,得一次茯苓渣;
一次茯苓渣水煮,烘干滤渣,得二次茯苓渣;
二次茯苓渣用碱液浸提,过滤除去滤渣,所得滤液加酸调pH、静置,得沉淀物;
沉淀物用纯水脱盐后烘干,得碱溶性茯苓多糖。
7.根据权利要求1所述的茯苓多糖水凝胶的制备方法,其特征在于:所述碱溶性茯苓多糖分散液的浓度为1wt%±0.1wt%、pH=7±0.2。
8.一种茯苓多糖水凝胶,其特征在于:通过权利要求1~7任一项所述的茯苓多糖水凝胶的制备方法制备得到。
9.权利要求8所述的茯苓多糖水凝胶在制备缓释药物中的用途。
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