CN114569548A - 一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法 - Google Patents
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法 Download PDFInfo
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Abstract
本发明公开了一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法，由以下步骤制成：(1)纺丝液的制备；(2)静电纺丝纤维的制备；(3)口腔溃疡含漱液的制备。本发明采用的静电纺丝纤维结构多孔交织，该结构通过模拟人体中的ECM为细胞膜上的受体提供粘附点从而促进伤口愈合，除此之外，药效明确的中药单体吸附在静电纺丝纤维上使药物释放更加迅速，显著提高生物利用度。本发明利用静电纺丝纤维技术将两者结合，最终获得能在口腔溃疡表面形成保护膜从而遮蔽口腔溃疡伤口的一种含漱液，该含漱液抗菌性能极佳，有较好的生物相容性，能够更快地在口腔溃疡创面上成膜，加快损伤修复，疗效更优。
Description
技术领域
本发明涉及口腔含漱液制备领域，具体涉及一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法。
背景技术
口腔溃疡是一种常见的口腔黏膜疾病，发病率较高，发病原因与患者自身免疫功能出现紊乱、妇女妊娠期、某些药物的过多使用、感染、内分泌失调、身体其他系统疾病等有关。该病具有周期性、复发性等特点，多发生于患者的颊、唇以及舌缘等部位，当患者进食的时候溃疡部位会有剧烈的灼烧感，严重影响患者的日常生活。目前，临床上多使用激素类药物进行治疗，这类药物副作用大，极易刺激患者胃肠道。如果长期使用，会引起一系列的并发症，如十二指肠溃疡、消化道出血、骨质疏松、肌肉萎缩、伤口愈合延迟、高血压和动脉粥样硬化。
随着技术的发展，静电纺丝纤维技术逐渐用于医学领域，能够用于口腔溃疡创面修复。该技术可生产直径范围从纳米到微米的连续聚合物纤维，所得到的静电纺丝纤维具有良好的皮肤适用性。静电纺丝装置包括高压电源、注射针及静电纺丝纤维接收器。将高压施加到纺丝液上，从而在喷嘴尖端处形成泰勒锥。通过改变纺丝液流速使喷出的液体射流固化为不同性质的纤维，这些纤维在接收器中移动一段时间后得到静电纺丝纤维膜，具体过程见图1。和普通的膜质相比，这种静电纺丝纤维是一种更为优良的伤口敷料，结构多孔交织，该结构可以模拟人体ECM结构从而给细胞膜上的受体提供粘附点，促进伤口的动态愈合。此外，将药理作用清晰明确的中药单体溶解于纺丝液中，药物将以微粒形式吸附在静电纺丝纤维上，该纤维具有较高的比表面积，能够控制药物释放的速度，大幅度提高了药物的生物利用度。目前，含漱液的主要作用是抑菌，刺激性较强，本方案所提供的一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法运用静电纺丝纤维技术，使含漱液在口腔溃疡伤口创面形成载有药物的物理保护膜，减少疼痛，中药活性成分在静电纺丝纤维膜上释放，各种中药单体作用于不同靶点而产生协同增效作用，能够更加快速治疗口腔溃疡，增加患者依从性。
发明内容
本发明的目的是为了克服现有技术的不足，提供一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法。本发明的另一个目的在于该含漱液可缓解口腔溃疡导致的疼痛。本发明的创新之处在于运用静电纺丝技术制备口腔含漱液，目前尚未有该应用。本发明的另外创新之处在于该中药配方疗效优良，和普通的中药制剂相比，本发明采用的为中药单体，浓度更高，药理作用明确。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法，其特征在于，包括如下步骤：
(1)纺丝液的制备：将纺丝溶质加入到有机溶剂中，溶解完全后加入中药单体组合物，即得纺丝液。其中，纺丝溶质与有机溶剂的质量与体积比为9-13％，中药单体组合物与有机溶剂的质量与体积比为0.5％-0.9％，纺丝溶质为聚丙烯腈(PAN)、聚乙烯醇(PVA)、聚苯乙烯(PS)中任意一种或两种的组合，有机溶剂为N,N-二甲基甲酰胺(DMF)、乙酰基二甲胺(DMAC)、二甲基亚砜(DMSO)中一种或两种的组合，中药单体组合物由鱼腥草素15-30份、栀子苷12-23份、绿原酸8-14份、木犀草素16-30份、甲基丁香酚9-17份组成。
(2)静电纺丝纤维的制备：将步骤(1)中的纺丝液进行静电纺丝，其中静电纺丝机电喷条件为：电压15-30kV，接收距离15-20cm，注射器的推进速率为0.8mL/h，环境温度为25-35℃，湿度为40-50％。当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加至纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
本方案所提供的含漱液制备方法，通过静电纺丝法将含有中药单体组合物的纺丝液加工成口腔含漱液，该含漱液可在口腔黏膜创面上形成纳米纤维膜，该膜具有一定细胞相容性，还可以发挥持久抗菌效果，并且加入了卡波姆，卡波姆粘附性力强，对糖蛋白寡糖链上的糖残基有亲和力，能够与口腔黏膜糖蛋白相互缠绕，可加速伤口愈合。卡波姆和口腔黏膜糖蛋白形成了粘液凝胶网状结构之后与含有中药单体组合物的纳米纤维一同在口腔溃疡的伤口创面上形成物理遮蔽膜。在实际使用本产品进行含漱时，含漱液在口腔内逐渐形成一种半固体的流体膜，粘附在伤口面上，阻隔唾液或唾液中病原微生物对黏膜创口的侵蚀与感染，缓解口腔创面的疼痛，并且能释放药物进行治疗。又由于本产品采用药用辅料，无代谢毒性且有较好的生物相容性，所制口腔溃疡含漱液能缓解口腔溃疡及口腔炎症所带来的疼痛，安全有效，并具有较大的临床应用价值。此外，本方案运用的中药配方优良，充分发挥药物的协同作用及中医药多种有效成分多靶点作用。
鱼腥草素具有抗菌、抗病毒、提高机体免疫力、利尿等作用，对卡他球菌、溶血性链球菌、流感杆菌、肺炎双球菌和金黄色葡萄球菌有显著的抑制作用，对大肠杆菌、痢疾杆菌、伤寒杆菌及孢子丝菌等也有抑制作用。
栀子苷能抗炎镇痛、保护心脑血管，有效增强细胞内抗氧化体系的活性，调节细胞内一氧化氮合酶活性，促进一氧化氮生成恢复正常水平从而达到抗炎镇痛和保护心脑血管的效果。
绿原酸是由咖啡酸的1位羧基和奎尼酸的3位羟基缩合成酯的天然产物，具有抗菌、抗炎作用，能够破坏肺炎链球菌、金葡球菌和志贺氏菌的细胞膜，增加外膜通透性使细菌的屏障功能丧失从而发挥抗菌活性；通过清除细胞内的ROS，抑制上调核因子信号通路从而较少白介素-8的产生，最终发挥抗炎作用。
木犀草素具有抗病毒、抗菌、抗炎等作用，能够抑制呼吸道合胞病毒，直接作用于STAT1的负调节因子COCS1靶点从而抑制病毒的复制；通过抑制细胞内DNA拓扑异构酶的活性使细胞凋亡，能够有效减少假丝酵母、表面葡萄球菌、大肠杆菌、粪肠球菌和变异链球菌等多种口腔致病菌在生物膜上的形成和粘附，从而促进口腔创面愈合；通过抑制HMGB1-NF-κB信号通路发挥抗炎活性。
甲基丁香酚具有镇痛、损伤修复的作用，通过作用于门冬氨酸受体影响细胞兴奋传导，发挥镇痛作用；通过调节多种信号通路发挥神经系统、胃肠道的保护作用，抑制细胞凋亡，改善和减少脑梗死体积，促进损伤黏膜修复。除此之外，甲基丁香酚治疗肿瘤化疗并发的口腔溃疡效果好，能够有效减短口腔溃疡愈合时间，缓解疼痛。
附图说明
图1为静电纺丝膜制备过程，其中a为静电纺丝原理图，b为静电纺丝纤维膜示意图，c为静电纺丝纤维形貌特征图。
图2为本发明实施例1的静电纺丝纤维形貌图
图3为本发明实施例2的静电纺丝纤维形貌图。
图4为本发明实施例3的静电纺丝纤维形貌图。
图5为本发明实施例4的静电纺丝纤维形貌图。
图6为本发明实施例5的静电纺丝纤维形貌图。
图7为本发明实验中各药液对金黄色葡萄球菌抑菌效果图，其中1为含漱液，2为西瓜霜喷剂，3为生理盐水。
图8为本发明实验中各药液对牙龈卟啉单胞菌抑菌效果图，其中1为含漱液，2为西瓜霜喷剂，3为生理盐水。
图9为本发明实验中各组SD大鼠口腔溃疡面积变化图。
具体实施方式
下面结合具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法如下：
(1)纺丝液的制备：将聚丙烯腈(PAN)加入到N,N-二甲基甲酰胺(DMF)中，溶解完全后加入中药单体组合物，即得纺丝液。其中，PAN与DMF的质量与体积比为9％，中药单体组合物与DMF的质量与体积比为0.5％，中药单体组合物由鱼腥草素20份、栀子苷15份、绿原酸9份、木犀草素17份、甲基丁香酚16份组成。
(2)静电纺丝纤维的制备：注射器吸取纺丝液，然后将注射器安装在推进泵上，针头与接收装置之间连接高压电源，接通电源后进行静电纺丝，其中，电压20V，接收距离18cm，注射器的推进速率为0.8mL/h，环境温度为25℃，湿度为40％。当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维，所得静电纺丝纤维形貌见图2。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加至纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
实施例2
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法如下：
(1)纺丝液的制备：将聚乙烯醇(PVA)加入到乙酰基二甲胺(DMAC)中，溶解完全后加入中药单体组合物，即得纺丝液。其中PVA与DMAC的质量与体积比为10％，中药单体组合物与DMAC的质量与体积比为0.6％，中药单体组合物由鱼腥草素30份、栀子苷16份、绿原酸13份、木犀草素25份、甲基丁香酚16份组成。
(2)静电纺丝纤维的制备：注射器吸取纺丝液，然后将注射器安装在推进泵上，针头与接收装置之间连接高压电源，接通电源后进行静电纺丝，其中，电压25V，接收距离18cm，注射器的推进速率为0.8mL/h，环境温度为25℃，湿度为45％。当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维。所得静电纺丝纤维形貌见图3。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加至纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
实施例3
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法如下：
(1)纺丝液的制备：将聚苯乙烯(PS)加入到由二甲基亚砜(DMSO)与N,N-二甲基甲酰胺(DMF)组成的混合溶液中，完全溶解后加入中药单体组合物，即得纺丝液。其中DMSO与DMF的比例为2:3，PS与混合溶液的质量与体积比为11％，中药单体组合物与混合溶液的质量与体积比为0.7％，中药单体由鱼腥草素16份、栀子苷23份、绿原酸10份、木犀草素17份、甲基丁香酚11份组成。
(2)静电纺丝纤维的制备：注射器吸取纺丝液，然后将注射器安装在推进泵上，针头与接收装置之间连接高压电源，接通电源后进行静电纺丝，其中，电压30V，接收距离20cm，注射器的推进速率为0.8mL/h，环境温度为25℃，湿度为50％。当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维。所得静电纺丝纤维形貌见图4。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
实施例4
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法如下：
(1)纺丝液的制备：将聚丙烯腈(PAN)与聚乙烯醇(PVA)加入到N,N-二甲基甲酰胺(DMF)中，溶解完全后加入中药单体组合物，即得纺丝液。其中PAN与PVA的比例为1:3，混合溶质与DMF的质量与体积比为11％，中药单体组合物与DMF的质量与体积比为0.8％，中药单体组合物由鱼腥草素28份、栀子苷22份、绿原酸14份、木犀草素27份、甲基丁香酚16份组成。
(2)静电纺丝纤维的制备：注射器吸取纺丝液，然后将注射器安装在推进泵上，针头与接收装置之间连接高压电源，接通电源后进行静电纺丝，其中，电压15V，接收距离18cm，注射器的推进速率为0.8mL/h，环境温度为25℃，湿度为45％，当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维。所得静电纺丝纤维形貌见图5。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加至纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
实施例5
一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法如下：
(1)纺丝液的制备：将聚乙烯醇(PVA)加入到N,N-二甲基甲酰胺(DMF)中，溶解完全后加入中药单体组合物，即得纺丝液。其中PVA与DMF的质量与体积比为12％，中药单体组合物与DMF的质量与体积比为0.8％，中药单体组合物由鱼腥草素23份、栀子苷13份、绿原酸14份、木犀草素24份、甲基丁香酚16份组成。
(2)静电纺丝纤维的制备：注射器吸取纺丝液，然后将注射器安装在推进泵上，针头与接收装置之间连接高压电源，接通电源后进行静电纺丝，其中，电压30V，接收距离17cm，注射器的推进速率为0.8mL/h，环境温度为25℃，湿度为50％，当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维。所得静电纺丝纤维形貌见图6。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加至纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
为了研究实施例1中制备的一种含有静电纺丝纤维的中药口腔溃疡含漱液治疗作用，具体实验如下：
一.口腔含漱液抑菌效果
1.实验材料
用实施例1中方法所制含漱液、MH琼脂培养基(购自广东环凯生物科技有限公司)、血平板(购自广东环凯生物科技有限公司)、高压灭菌锅(购自广州越特科学仪器有限公司)、生化培养箱(购自广州市深华生物技术有限公司)、三金桂林西瓜霜喷剂(购自广东大参林连锁药店有限公司)、牙龈卟啉单胞菌(购自北京百欧博伟生物技术有限公司)、金黄色葡萄球菌(购自中国食品发酵工业研究院有限公司)。
2.实验原理
金黄色葡萄球菌及牙龈卟啉单胞菌是口腔疾病中的主要致病菌，本实验采取体外平皿打孔法观察该含漱液抑菌效果。
3.操作步骤
将标准菌株由低温冰箱中取出，恢复至室温，无菌条件下将菌株置于36℃环境下培养36h后划线接种于培养基中。待培养完成，将金黄色葡萄球菌与牙龈卟啉单胞菌进行鉴定后，用接种环挑取菌落，使其悬浮于生理盐水中，充分振荡。采用平皿打孔法，将MH琼脂培养基高压灭菌后倾注平板，冷却后将菌液均匀涂布至培养基表面。用打孔器在平皿均匀打孔3个，每个孔内加入30μL药液，分别为含漱液、西瓜霜喷剂、生理盐水。12h后用游标卡尺测量抑菌环直径，以抑菌环边缘以见不到细菌明显生长为限，测量完全抑制的区域直径。
4.实验结果
采用SPSS21.0统计学软件处理数据，计量资料用x-±s表示，用t检验，p＜0.05为差异具有统计学意义。如表1、图7、图8所示，本发明所制含漱液对金黄色葡萄球菌、牙龈卟啉单胞菌均有较好的抑菌效果，与西瓜霜喷剂相比，抑菌效果更强。
表1含漱液对金黄色葡萄球菌及牙龈卟啉单胞菌抑菌环直径分析(n＝6，x-±s，mm)
注：与西瓜霜喷剂对照组相比较，*P＜0.01。
二、口腔溃疡治疗效果
1.实验材料
SD大鼠48只，体重200g-210g，雌雄各半(购自广东省医学实验动物中心)，NaOH晶体(购自广州市锦天化工科技有限公司)，水合氯醛(购自深圳市星凯越生物科技有限公司)，含漱液，三金桂林西瓜霜喷剂(购自广东大参林连锁药店有限公司)。
2.实验原理
制备大鼠口腔溃疡模型，用棉签蘸取西瓜霜喷剂药液与含漱液涂敷在大鼠口腔创面上，观察创面面积变化。
3.操作步骤
使用水合氯醛麻醉大鼠，用镊子取NaOH晶体在大鼠下切牙区唇侧牙龈处灼烧60s，随后用生理盐水清洗，形成直径15mm的创面。将48只大鼠随机分3组，A组为实验组，B组为阳性药物对照组，C组为空白对照组。A组每日用棉签蘸取含漱液涂敷创面两次，早晚各1次，涂敷后30min不进食，连续7天。B组每日用棉签蘸取西瓜霜喷剂药液涂敷创面两次，早晚各1次，涂敷后30min不进食，连续7天。C组每日用棉签蘸取生理盐水涂敷创面两次，早晚各1次，涂敷后30min不进食，连续7天。用游标卡尺测量各大鼠口腔溃疡面积，计算溃疡面积＝0.25×D1×D2×圆周率(约3.14)(游标卡尺测量溃疡最大横径D1，最大纵径D2)。
4.实验结果
采用SPSS21.0统计学软件处理数据，计量资料用x-±s表示，用t检验，p＜0.05为差异具有统计学意义。由表2、图9可见，A组和B组均能促进大鼠口腔溃疡伤口的修复，与西瓜霜喷剂药液相比，使用本发明所制含漱液的大鼠口腔溃疡面积变化率更大，说明本发明所制含漱液修复力更强，创面能够更快愈合。
表2各组SD大鼠口腔溃疡面积变化情况(x-±s，mm2)
注：与C组比较，*P＜0.05，**P＜0.01；与B组比较，#P＜0.05。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (4)
1.一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法，其特征在于，包括如下步骤：
(1)纺丝液的制备：将纺丝溶质加入到有机溶剂中，溶解完全后加入中药单体组合物，即得纺丝液。其中，纺丝溶质与有机溶剂的质量与体积比为9-13％，中药单体组合物与有机溶剂的质量与体积比为0.5％-0.9％，纺丝溶质为聚丙烯腈(PAN)、聚乙烯醇(PVA)、聚苯乙烯(PS)中任意一种或两种的组合，有机溶剂为N,N-二甲基甲酰胺(DMF)、乙酰基二甲胺(DMAC)、二甲基亚砜(DMSO)中任意一种或两种的组合，中药单体组合物由鱼腥草素、栀子苷、绿原酸、木犀草素、甲基丁香酚组成。
(2)静电纺丝纤维的制备：将步骤(1)中的纺丝液进行静电纺丝，其中静电纺丝机电喷条件为：电压15-30kV，接收距离15-20cm，注射器的推进速率为0.8mL/h，环境温度为25-35℃，湿度为40-50％。当接收装置上的静电纺丝膜收集完毕后，取出静电纺丝膜打碎得到静电纺丝纤维。
(3)口腔溃疡含漱液的制备：将静电纺丝纤维添加至纯化水中，添加的静电纺丝纤维与纯化水之间的质量与体积比为12％，添加麦芽糖糊精、卡波姆、吐温80、丙二醇、甘油、木糖醇等辅料即得含漱液。
2.如权利要求1所述的一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法，其特征在于，步骤(1)中的中药单体组合物由以下重量份的单体组成：称取鱼腥草素15-30份、栀子苷12-23份、绿原酸8-14份、木犀草素16-30份、甲基丁香酚9-17份，混合均匀即得。
3.如权利要求1所述的一种含有静电纺丝纤维的中药口腔溃疡含漱液的制备方法，其特征在于，步骤(2)中具体包括：从接收装置上取下静电纺丝纤维膜，置于10000rpm/min的高速搅拌器中搅拌，将静电纺丝膜纤维全部打碎。
4.如权利要求1所述的一种含有静电纺丝纤维的中药口腔溃疡含漱液在缓解口腔溃疡疼痛中的应用。
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