CN107189082B - 白芨壳聚糖基温敏水凝胶的制备方法 - Google Patents
白芨壳聚糖基温敏水凝胶的制备方法 Download PDFInfo
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Abstract
本发明提出了一种白芨壳聚糖基温敏水凝胶的制备方法，以白芨水提物、壳聚糖、甘油磷酸钠为原料；所得产品同时兼具了壳聚糖和白芨的特性，具有良好的抑菌作用及良好的生物相容性，作为新型创伤敷料有良好的研究应用前景。
Description
技术领域
本发明涉及医药材料领域，特别是涉及白芨壳聚糖基温敏水凝胶的制备方法。
背景技术
壳聚糖（chitosan,CS）是一类由2-氨基-2-脱氧-葡萄糖通过1,4-糖苷键连结的带正电荷的直链多糖，是目前自然界唯一大量存在的碱性多糖。在外观上呈白色或淡黄色半透明片状固体，有珍珠光泽; 不溶于水及碱性溶液，可溶于酸性溶液; 其来源广泛，无毒无害，具有良好的黏附作用和组织相容性，易于生物降解; 具有良好的保湿性、润湿性，并能防止产生静电等。壳聚糖在医学、药学等领域得到了深入的研究和广泛的应用。白芨有收敛止血，消肿生肌的功效。制备白芨温敏水凝胶，同时兼具了壳聚糖和白芨的特性，在生物工程和生物制药领域有广阔的应用前景。
授权公告号CN 104984401 A（申请号201510434128.3）的中国专利文献公开了一种温敏水凝胶/磷酸三钙材料的制备方法，该方法步骤流程包括将温敏水凝胶在低温下溶解、按一定比例加入磷酸三钙粉体、混合材料随意塑形成任何想要的形状。公布号CN103004757 A（申请号201210571657.4）的中国专利文献公开了一种多孔温敏水凝胶缓释剂及其制备方法。本发明以聚N-异丙基丙烯酰胺多孔温敏水凝胶为载体，在凝胶内负载上药物，利用聚N-异丙基丙烯酰胺的温度敏感特性，来实现对药物的控制释放。当温度低于水凝胶的LCST时，药物释放很慢，而当温度高于其LCST时，释放速率加快。
发明内容
针对现有技术的问题，本发明的目的是提供一种白芨壳聚糖基温敏水凝胶的制备方法，白芨壳聚糖基温敏水凝胶具有良好的抑菌作用及良好的生物相容性以及无纤毛毒性，作为新型创伤敷料有良好的研究应用前景。
本发明的技术方案是：
白芨壳聚糖基温敏水凝胶的制备方法，以白芨水提物、壳聚糖、甘油磷酸钠为原料，具体步骤如下：
（1）取白芨洗净，用纱布包好放于蒸馏水中浸泡30分钟，用电阻炉功率调制600W,大火进行性加热30分钟，后用400W功率小火继续加热，然后过滤除去滤渣得白芨提取液备用；
（2）取壳聚糖粉碎研磨5-15分钟（优选的：粉碎研磨10分钟），置于0.1mol/L的冰乙酸中充分溶解后，加入白芨提取液，用pH计测量其pH值，逐滴添加56%的甘油磷酸钠，并不断搅拌，用饱和的磷酸氢钠溶液调节pH至6.96；
（3）将上述配置好的壳聚糖溶液置于容器中，放入恒温水浴锅中加热，待溶液不流动时，即形成白芨壳聚糖基温敏水凝胶。
所述的温敏水凝胶的制备方法，壳聚糖：白芨水提物：甘油磷酸钠的质量比为4:5-20:20（优选的：质量比为2:5:10）。
所述的温敏水凝胶的制备方法，步骤（1）蒸馏水的用量为白芨重量的20倍。
所述的温敏水凝胶的制备方法，步骤（1）小火继续加热25-35分钟（优选的：小火继续加热30分钟）。
所述的温敏水凝胶的制备方法，步骤（2）冰乙酸水溶液的用量为壳聚糖质量的50倍。
所述的温敏水凝胶的制备方法，步骤（3）恒温水浴锅的温度为34-40℃（优选的：恒温水浴锅的温度为37℃）。
本发明提供的白芨壳聚糖基温敏水凝胶的制备方法科学，工艺简单，所得产品同时兼具了壳聚糖和白芨的特性，具有良好的抑菌作用及良好的生物相容性以及无纤毛毒性，作为新型创伤敷料有良好的研究应用前景。
附图说明
图1：纤毛毒性试验的观察结果；
图2：为不同浓度的白芨壳聚糖基温敏水凝胶对NIH-3T3细胞存活率的影响结果；注：A1；A2;A3;A4;A5;A6依次代表稀释倍数，分别为是0倍、5倍、10倍、20倍、50倍、100倍，B.C.D.同上；柱状图横纵坐标分别为稀释倍数和细胞相对增值率；
图3：a所得温敏水凝胶相转变前；b所得温敏水凝胶相转变后。
具体实施方式
下面结合实施例和实验例详细说明本发明的技术方案，但保护范围不限于此。
本申请所用材料均可从市场购买，其中白芨购自聊城市中医院；壳聚糖购自浙江金壳生物化学有限公司，批号为20140318C；甘油磷酸钠为国产分析纯，购自湖北远成赛创科技有限公司。
实施例1白芨壳聚糖基温敏水凝胶的制备方法，步骤如下：
（1）取白芨4g洗净，用纱布包好放于80ml蒸馏水中浸泡30分钟，用电阻炉功率调制600W,大火进行性加热30分钟，后用400W功率小火继续加热25分钟，然后过滤除去滤渣得白芨提取液备用；
（2）取壳聚糖0.2g粉碎研磨5分钟，置于10ml，0.1mol/L的冰乙酸中充分溶解后，加入白芨提取液0.25ml，用pH计测量其pH值，逐滴添加56%的甘油磷酸钠1.0ml，并不断搅拌，用饱和的磷酸氢钠溶液调节pH至6.96；
（3）将上述配置好的壳聚糖溶液置于容器中，放入34℃恒温水浴锅中加热，待溶液不流动时，即形成白芨壳聚糖基温敏水凝胶。
实施例2白芨壳聚糖基温敏水凝胶的制备方法，步骤如下：
（1）取白芨4g洗净，用纱布包好放于80ml蒸馏水中浸泡30分钟，用电阻炉功率调制600W,大火进行性加热30分钟，后用400W功率小火继续加热35分钟，然后过滤除去滤渣得白芨提取液备用；
（2）取壳聚糖0.2g粉碎研磨15分钟，置于10ml，0.1mol/L的冰乙酸中充分溶解后，加入白芨提取液1.0ml，用pH计测量其pH值，逐滴添加56%的甘油磷酸钠1.0ml，并不断搅拌，用饱和的磷酸氢钠溶液调节pH至6.96；
（3）将上述配置好的壳聚糖溶液置于容器中，放入40℃恒温水浴锅中加热，待溶液不流动时，即形成白芨壳聚糖基温敏水凝胶。
实施例3白芨壳聚糖基温敏水凝胶的制备方法，步骤如下：
（1）取白芨4g洗净，用纱布包好放于80ml蒸馏水中浸泡30分钟，用电阻炉功率调制600W,大火进行性加热30分钟，后用400W功率小火继续加热30分钟，然后过滤除去滤渣得白芨提取液备用；
（2）取壳聚糖0.2g粉碎研磨10分钟，置于10ml，0.1mol/L的冰乙酸中充分溶解后，加入白芨提取液0.50ml，用pH计测量其pH值，逐滴添加56%的甘油磷酸钠1.0ml，并不断搅拌，用饱和的磷酸氢钠溶液调节pH至6.96；
（3）将上述配置好的壳聚糖溶液置于容器中，放入37℃恒温水浴锅中加热，待溶液不流动时，即形成白芨壳聚糖基温敏水凝胶。
试验例1本发明所得白芨壳聚糖基温敏水凝胶的抑菌性能测定：
设置四组，其中A、B、C组分别对应实施例1-3中的白芨壳聚糖基温敏水凝胶，D组为不含白芨的壳聚糖温敏水凝胶；
在实验条件下，将保存的大肠杆菌进行活化培养，培养基为牛肉膏蛋白胨培养基，待培养基长出单个菌落后，用接种环挑取单个菌落制备大肠杆菌的菌悬液。无菌条件下，在90mm的培养基中加入适量的牛肉膏蛋白胨培养基，凝固后向培养基加入0.1ml菌悬液(107cfu/ml)，用涂布器涂布均匀。选取直径0.4cm打孔器，对凝胶样品进行打孔，制得直径0.4cm的圆柱体。在已经划好区域的培养基上，依次放置A、B、C、D，四种待测样品，待培养皿在37℃恒温培养箱中培养24h后，测量各个样品抑菌圈直径大小并记录数据。以同样的方法测试样品对金黄色葡萄球菌、肺炎球菌、绿脓杆菌的抑菌效果，同时测量各个样品抑菌圈直径大小并记录数据。
试验结果如下表所示：
表1：白芨壳聚糖基温敏水凝胶抑菌试验
通过表格1中实验组（ABC组）与空白组（D组）对照，明显看出白芨壳聚糖基温敏水凝胶对于四种菌具有较好的接触抑菌的作用，各样品与培养基的接触面均没有细菌的生长。同时发现随着白芨含量的增加，抑菌圈的直径明显的增加，可以体现抑菌的作用效果明显增强。
试验例2本发明所得白芨壳聚糖基温敏水凝胶的纤毛毒性实验：
2.1 蟾蜍上颚粘膜观察
本试验中，选用无纤毛毒性的生理盐水为阴性对照样品，有严重纤毛毒性的去氧胆酸钠为阳性对照品，分别评价不同浓度的试验样品的纤毛毒性；共分为5组，分别为A组（实施例1的水凝胶），B组（实施例2的水凝胶），C组（实施例3的水凝胶），D组（生理盐水组），E组（1%去氧胆酸钠组）。
将30只蟾蜍平均分成5组，将蟾蜍仰卧固定于蛙板上,使口腔张开,以止血钳牵拉,防止闭合和吞咽。分别将0.5mL水凝胶,0.5mL去氧胆酸钠应用到蟾蜍上颚部位,以生理盐水0.5mL作为对照,保留30min。用眼科小剪刀从口腔上剪下3-4 mm2粘膜,用生理盐水将表面的血液和粘液漂洗干净,并将其平铺于载玻片上,滴加生理盐水,轻轻盖上盖玻片,于10*40倍倒置显微镜下观察黏膜形态。
试验结果如附图1所示：
取下粘膜后立即在显微镜下观察，由图片1结果显示可知，生理盐水组的粘膜表面和纤毛清晰完整，纤毛活动活跃，呈流水状摆动; 1%去氧胆酸钠组纤毛停止摆动，粘膜受损严重，表面杂乱，纤毛基本脱落，只见裸露基部，周围可见很多脱落细胞; 其他药物组粘膜和纤毛较完整清晰，无脱落等异常现象，纤毛运动较活跃。
2.2纤毛持续运动时间和持续运动百分率的测定
将黏膜置于预先用少量蒸馏水饱和的层析缸中，密闭，环境温度25℃，此后每隔一定的时间取出上颚标本于图像分析系统下观察，若蟾蜍纤毛继续运动则放回层析缸中，直至纤毛运动停止。同时记录从给药开始至纤毛停止运动的时间，即蟾蜍纤毛的摆动时间（Persistent Vibration Duration,PVD）。以各给药组的PVD除以生理盐水对照组的PVD即得到纤毛持续运动的百分率（Percentage of Persistent Vibration ,PPV)。百分率越低表示药物对于纤毛的毒性越大，百分率越高则表示药物对纤毛的毒性越小。
试验结果如表2所示：
表2各组纤毛持续运动时间和持续运动百分率（x±s，n=6）
由表2可知，与生理盐水组比较，去氧胆酸钠组纤毛持续运动时间短于生理盐水组，有极显著性差异。同时给药组的纤毛持续运动时间也短于生理盐水组，但是无显著性差异，可认为给予的药物对纤毛运动无影响。用生理盐水冲洗粘膜后，除去氧胆酸钠组的纤毛之外，其余药物组纤毛均恢复摆动。此结果提示白芨壳聚糖基温敏水凝胶的对蟾蜍口腔黏膜纤毛运动基本没影响，因此水凝胶对蟾蜍纤毛毒性符合生物材料的安全评价标准。
2.3实验样品纤毛输送速率的测定
分组、给药同实验2.1的方法。给药5 min后在上腭靠前方部位用毛细管放置甲基红颗粒，此时颗粒将沿黏膜表面缓慢向咽部移动留下红色轨迹，用秒表记录颗粒移动1cm所需时间，每只蟾蜍测定3次，取均值后计算纤毛输送速率。
上述实验的统计学处理方法，计量资料数据以`x± s表示，应用SPSS19.0系统分析软件进行分析，采用单因素方差分析及SNK检验，P＜0.05为差异有统计意义。
试验结果如表3所示：
表3对纤毛的输送速率的影响（x±s，n=6）
如表3所示，所示各给药组与生理盐水组纤毛输送速率比较，A、B、C组（凝胶组）与生理盐水对照组无显著差异（P＜0.05），其余1%去氧胆酸钠组（E组）与生理盐水组差异有显著性（P>0.05），表明白芨壳聚糖基温敏水凝胶对于纤毛的传输速率基本没有影响。
试验例3本发明所得白芨壳聚糖基温敏水凝胶的细胞相容性试验：
3.1 不同白芨含量的壳聚糖基温敏水凝胶的48h的制备
各种样品水凝胶分别按照样品:细胞培养液=0.2g:1ml的比例予以浸泡48h得试验样品的浸提液，0.2um的滤膜推虑除菌，将推虑好的试验样品进行梯度稀释，依次是0倍、5倍、10倍、20倍、50倍、100倍的样品提取液。
3.2 细胞的培养
小鼠成纤维细胞（NIH-3T3），细胞培养在添加,15％胎牛血清的DMEM培养基中，培养条件为：5％CO2、37℃、饱和湿度CO2培养箱中，采用倒置相差显微镜下观察细胞形态。
3.3 细胞存活率的测定
将处于对数生长期的细胞按4000个/孔种植到96孔板，37℃、5％CO2培养，待NIH-3T3细胞长到80％以上，除正常组外所有处理组，分别经不同浓度的分别处理细胞24h后，取出96孔板，甩出细胞培养液；每孔添加100ul,10％三氯乙酸（TCA）放置4℃恒温冰箱固定1h；甩出TCA，用超净水对每孔侧壁冲洗5遍，放烘箱37℃烘干；每孔添加50ul的0.4％SRB染料并放置摇床5min,吸出染料；1％的乙酸冲洗5遍并放置烘箱烘干；每孔添加100ul,100 mmol/LTris碱溶解；492nm处测OD吸光值。计算细胞的相对增值率（relative growth rate,RGR）*100％。
根据美国药典中细胞毒性的六级标准评价材料的毒性大小，细胞毒性反应分级如下：
表4细胞相对增值率与细胞毒性分级的关系
毒性的判定标准:0 级和Ⅰ级判为合格，Ⅱ级者结合形态分析综合评价，Ⅲ～Ⅳ级者判为不合格。
采用SPSS19.0软件包统计分析,计量资料以“`x ± s”表示，采用t检验进行统计学分析。*P＜0.05 为差异显著，**P＜0.01为差异极显著。细胞存活率 (％ )=(处理组 OD值／对照组 OD值 )X 100％，数据采用采用SPSSvl1.5软件包统计分析。
试验结果如附图2所示：
不同浓度的白芨壳聚糖基温敏水凝胶对NIH-3T3细胞存活率的影响如附图2所示，不同浓度稀释倍数的白芨壳聚糖基温敏水凝胶复合物以及壳聚糖温敏水凝胶的浸提液对于NIH-3T3细胞都没有表现出细胞毒性。水凝胶提取液稀释50倍，100倍对于NIH-3T3细胞的增值有着明显的促进作用（P＜0.01)。浸提液稀释5倍、10倍、20倍，对于细胞的生长基本没有影响。浸提液稀释0倍，细胞的增值率相对降低，但是对于NIH-3T3细胞造成不了毒性作用。因此水凝胶浸提液对于NIH-3T3细胞毒性符合生物材料的安全评价标准。
综上可知，本发明白芨壳聚糖基温敏水凝胶对于大肠杆菌、金黄色葡萄球菌、绿脓杆菌、肺炎球菌均有较好的接触抑菌作用，并且随着水凝胶中白芨含量的提高抑菌效果也随之增强，同时与壳聚糖温敏水凝胶相比其抑菌作用更强；白芨壳聚糖基温敏水凝胶和壳聚糖温敏水凝胶24h浸提液对NIH-3T3细胞均没有毒性并且有良好的细胞相容性；白芨壳聚糖基温敏水凝胶的对蟾蜍口腔黏膜纤毛运动基本没影响。因此由于白芨壳聚糖基温敏水凝胶具有良好的抑菌作用及良好的生物相容性以及无纤毛毒性，作为新型创伤敷料有良好的研究应用前景。
且图3为所得温敏水凝胶相转变前后的状态图，其中a为所得温敏水凝胶相转变前；b为所得温敏水凝胶相转变后，由此可看出该温敏水凝胶可随温度的变化，发生相变，常温状态下为流动状态，体温状态下为不流动状态。
应当指出的是，具体实施方式只是本发明比较有代表性的例子，显然本发明的技术方案不限于上述实施例，还可以有很多变形。本领域的普通技术人员，以本发明所明确公开的或根据文件的书面描述毫无异议的得到的，均应认为是本专利所要保护的范围。
Claims (3)
1.白芨壳聚糖基温敏水凝胶的制备方法，其特征在于，以白芨水提物、壳聚糖、甘油磷酸钠为原料，壳聚糖：白芨水提物：甘油磷酸钠的质量比为2:5:10，具体步骤如下：
（1）取白芨洗净，用纱布包好放于蒸馏水中浸泡30分钟，蒸馏水的用量为白芨重量的20倍，用电阻炉功率调制600W,大火进行性加热30分钟，后用400W功率小火继续加热30分钟，然后过滤除去滤渣，得白芨提取液备用；
（2）取壳聚糖粉碎研磨10分钟，置于0.1mol/L的冰乙酸中充分溶解后，加入白芨提取液，用pH计测量其pH值，逐滴添加56%的甘油磷酸钠，并不断搅拌，用饱和的磷酸氢钠溶液调节pH至6.96；
（3）将上述配置好的壳聚糖溶液置于容器中，放入恒温水浴锅中加热，恒温水浴锅的温度为34-40℃，待溶液不流动时，即形成白芨壳聚糖基温敏水凝胶。
2.如权利要求1所述的制备方法，其特征在于，步骤（2）冰乙酸水溶液的用量为壳聚糖质量的50倍。
3.如权利要求1所述的制备方法，其特征在于，步骤（3）恒温水浴锅的温度为37℃。
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| CN103665400A (zh) * | 2013-11-12 | 2014-03-26 | 唐忠海 | 白芨止血凝胶的制备方法及应用 |
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| CN108939141B (zh) * | 2018-06-13 | 2021-05-18 | 上海长海医院 | 一种用于烧伤创面愈合的中药复合多孔支架材料及制备方法与应用 |
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