CN105878394A - 一种纳米银抗炎杀菌温敏水凝胶及其制备方法 - Google Patents
一种纳米银抗炎杀菌温敏水凝胶及其制备方法 Download PDFInfo
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- CN105878394A CN105878394A CN201610220822.XA CN201610220822A CN105878394A CN 105878394 A CN105878394 A CN 105878394A CN 201610220822 A CN201610220822 A CN 201610220822A CN 105878394 A CN105878394 A CN 105878394A
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Abstract
本发明公开了一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成：壳聚糖124‑140；硝酸银0.06‑0.08；甘油磷酸纳7‑11；丝素蛋白42‑60；丙烯酸酯28‑36，碳酸锌17‑21；赖氨酸8‑16；十大功劳根12‑20；荆芥穗9‑13；桉叶油12‑18；儿茶7‑13；细辛5‑12；防风6‑9。本发明的优良效果还表现在：本发明所得温敏水凝胶抗菌谱广、无细胞毒性，高效低毒；在药力渗透、透气性、促进伤口愈合等方面具有显著优势，且制备成本低、制备工艺简单等。
Description
技术领域
本发明涉及医药材料领域，特别是涉及一种纳米银抗炎杀菌温敏水凝胶及其制备方法。
背景技术
壳聚糖是自然界唯一大量存在的碱性多糖，是甲壳素经脱乙酰化后形成的一种阳离子多糖。壳聚糖广泛存在于节肢动物的外壳、真菌和酵母的细胞壁中，资源量非常丰富，同时具有可生物降解性、无毒、抑菌等特性。壳聚糖分子上具有大量的羟基和氨基，易被化学修饰，能与金属离子结合形成配合物，形成一系列新型壳聚糖衍生物，具有了更多的生物学功能，因此在医学、生物学领域得到了深入的研究和广泛的应用。
银离子在早期就由于其优异的抗菌杀菌性能被人们所广泛关注,其在医药材料和抗菌材料等领域具有广阔的应用前景。用低浓度银离子处理后的细胞没有明显的细胞聚集、细胞变形、细胞溶解等的变化。由此可见少量的银离子对人体没有明显的伤害作用,同时银具有很好的杀菌作用。纳米银是以纳米技术为基础研制而成的新型抗菌产品，比Ag +具有更稳定的物理化学特性，纳米银由于量子效应、小尺寸效应,不易产生耐药性，加速伤口的愈合，在医药材料和抗菌材料等领域具有广阔的应用前景。
温度敏感型水凝胶是指随着外界温度的变化而产生刺激响应性的智能材料。温度敏感型水凝胶结构中具有一定比例的亲水和疏水基团，温度的变化可以影响这些基团的疏水作用和大分子链间的氢键作用，从而影响其形态变化。最常见的温敏性水凝胶是聚Ｎ－异丙基丙烯酰胺水凝胶，这种水凝胶的体系内既有疏水基团又有亲水基团，这些基团可以在分子内与水分子作用，在分子外也与水分子发生作用。与合成高分子相比,天然高分子凝胶具有低毒性、生物相容性和可降解性等优点。温敏性壳聚糖水凝胶可根据温度变化处于不同物理形态。常温下为液态，进入人体组织后，在体温作用下凝胶化而固定在局部组织中。壳聚糖只在酸性环境下保持溶液状态，当pH值升高到 6.2时，壳聚糖溶液开始出现沉淀，若加入甘油磷酸钠，该系统可在中性条件下保持溶液状态，该系统具有温度敏感性，在体温以下保持溶液状态，温度升高到体温时可形成凝胶。它具有生物相容性好、体内降解时间及凝固时间可调节控制等优点, 在工程等领生物工程、医药领域已经得到广泛关注。
发明内容
本发明的目的在于提供一种纳米银抗炎杀菌温敏水凝胶及其制备方法，该温敏水凝胶对以大肠杆菌为代表的革兰氏阴性菌，以金黄色葡萄球菌为代表的革兰氏阳性菌，对以白色念珠菌为代表的真菌，均具有较好的抑菌作用，抗菌谱广、无细胞毒性，高效低毒；在药力渗透、透气性、促进伤口愈合等方面具有显著优势，且制备成本低、制备工艺简单等。
本发明的技术方案是：
一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成：壳聚糖124-140；硝酸银0.06-0.08；甘油磷酸纳7-11；丝素蛋白42-60；丙烯酸酯28-36，碳酸锌17-21；赖氨酸8-16；十大功劳根12-20；荆芥穗9-13；桉叶油12-18；儿茶7-13；细辛5-12；防风6-9。
优选的方案是：壳聚糖130-134；硝酸银0.065-0.075；甘油磷酸纳8-10；丝素蛋白48-54；丙烯酸酯30-34，碳酸锌18-20；赖氨酸10-14；十大功劳根14-18；荆芥穗10-12；桉叶油14-16；儿茶9-11；细辛7-10；防风7-8。
更加优选的：壳聚糖132；硝酸银0.07；甘油磷酸纳9；丝素蛋白50；丙烯酸酯32，碳酸锌19；赖氨酸12；十大功劳根16；荆芥穗11；桉叶油15；儿茶10；细辛8.5；防风7.5。
一种纳米银抗炎杀菌温敏水凝胶的制备方法，步骤如下：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖15-35分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡1-2小时（优选浸泡1.3-1.7小时），然后文火加热煎煮两次，每次各煎煮26-44分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨1-3小时（优选充分研磨1.5-2.5小时），紫外光照射4-8小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.05-0.15mol/L的醋酸水溶液溶解制成质量浓度为1.5%-2.5%的壳聚糖纳米银复合物溶液（优选制成质量浓度为1.7%-2.3%的壳聚糖纳米银复合物溶液），向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌20-50分钟（优选于室温下机械搅拌30-40分钟），然后经pH调节剂调节pH至6.2-7.0；
（5）将（4）所得的溶液放入35-38℃恒温水浴锅中（优选放入36-37℃恒温水浴锅中），3-6分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
上述温敏水凝胶的制备方法，优选的方案是，步骤（1）隔水炖20-30分钟（优选隔水炖25分钟）。
上述温敏水凝胶的制备方法，优选的方案是，步骤（1）每次各煎煮30-40分钟（优选每次各煎煮35分钟）。
上述温敏水凝胶的制备方法，优选的方案是，步骤（2）紫外光照射5-7小时（优选紫外光照射6小时）。
上述温敏水凝胶的制备方法，优选的方案是，步骤（3）用0.08-0.12mol/L的醋酸水溶液溶解（优选用0.1mol/L的醋酸水溶液溶解）。
上述温敏水凝胶的制备方法，优选的方案是，步骤（4）所述pH调节剂为磷酸氢二钠。
上述温敏水凝胶的制备方法，优选的方案是，步骤（4）调节pH至6.4-6.8（优选调节pH至6.6）。
各原料的药理功效或理化性质分别为：
壳聚糖：壳聚糖是甲壳素脱N-乙酰基的产物，一般而言，N-乙酰基脱去55%以上的就可称之为壳聚糖，或者说，能在1%乙酸或1%盐酸中溶解1%的脱乙酰甲壳素，这种脱乙酰甲壳素被称之为壳聚糖。生理活性：1、控制胆固醇；2、抑制细菌活性：壳聚糖在弱酸溶剂中易于溶解，特别值得指出的是溶解后的溶液中含有氨基（NH2+），这些氨基通过结合负电子来抑制细菌。壳聚糖的抑制细菌活性，使其在医药、纺织和食品等领域有着广泛的应用。3、预防和控制高血压；4、吸附和排泄重金属；5、免疫效果：壳聚糖具有更高的蛋白吸附能力；在降解酶（溶解酵素）的作用下，壳聚糖具降解性；壳聚糖很容易加工成线，适合做成线状或片状的医用材料；壳聚糖具有亲和力和溶解性，适用于生产各类衍生物；壳聚糖具有更高的化学活性；壳聚糖的持水性高；在血清中，壳聚糖易降解吸收；壳聚糖具有更高的生物降解性；壳聚糖表现出有选择性的高度抑制口腔链球菌生长的作用，同时并不影响其他有益细菌的生长。
硝酸银：无色透明斜方晶系片状晶体，易溶于水和氨水，溶于乙醚和甘油，微溶于无水乙醇，几乎不溶于浓硝酸。其水溶液呈弱酸性。硝酸银溶液由于含有大量银离子，故氧化性较强，并有一定腐蚀性，医学上用于腐蚀增生的肉芽组织，稀溶液用于眼部感染的杀菌剂。熔化后为浅黄色液体，固化后仍为白色。用途：分析化学用于沉淀氯离子，工作基准的硝酸银用于标定氯化钠溶液。无机工业用于制造其他银盐。电子工业用于制造导电粘合剂、新型气体净化剂、A8x分子筛、镀银均压服和带电作业的手套等。感光工业用于制造电影胶片、x光照相底片和照相胶片等的感光材料。电镀工业用于电子元件和其他工艺品的镀银，也大量用作镜子和保温瓶胆的镀银材料。电池工业用于生产银锌电池。医药上用作杀菌剂、腐蚀剂。日化工业用于染毛发等。分析化学中用于测定氯、溴、碘氰化物和硫氰酸盐。
甘油磷酸纳：为无色结晶或白色结晶状粉末；无臭，味咸。本品在水中易溶，在乙醇或丙酮中不溶。单纯的壳聚糖只在酸性环境下保持溶液状态,当pH值升高到6.2时,壳聚糖溶液开始出现沉淀,但在弱碱甘油磷酸钠参与下,该系统可在中性条件下保持稳定的溶液状态,这为该系统加载蛋白质类的药物甚至活的细胞提供了便利条件。最后是该系统具有温敏特性,在室温以下呈溶液状态,温度升高到体温时可固化形成凝胶,从而可以缓释加载药物的效果。
丝素蛋白：是从蚕丝中提取的天然高分子纤维蛋白，含量约占蚕丝的70%～80%，含有18种氨基酸，其中甘氨酸(Gly)、丙氨酸(Ala)和丝氨酸(Ser)约占总组成的80%以上。丝素本身具有良好的机械性能和理化性质，如良好的柔韧性和抗拉伸强度、透气透湿性、缓释性等，而且经过不同处理可以得到不同的形态，如纤维、溶液、粉、膜以及凝胶等。丝素蛋白具有很好的生物相容性，能制备成膜、凝胶、微胶囊等多种形态的材料，由于它独特的理化性能，丝素蛋白材料在生物医学材料领域被广泛的研究，如固定化酶材料、细胞培养基质、药物缓释剂、人工器官等。
丙烯酸酯：纯品为白色针状结晶。难溶于水和一般有机溶剂,能溶于热乙醇中,稍溶于热水中,易溶于稀酸、稀碱水溶液。在酸碱中稳定。丙烯酸类的聚合物具有一系列共同的特点，透明、低毒，易于配制，广泛的黏接性，耐水性，耐久性。使丙烯酸树脂在医用黏合剂中得到厂泛的应用。特性：①安全、可靠、无毒性、去三致（致癌、致畸、致突变）；②具有良好的生物相容性，不妨碍人体组织的自身愈合；③无菌，且可在一定时期内保持无菌；④在有血液和组织液的条件下可以使用；⑤在常温、常压下可以实现快速黏合；⑥具有良好的黏合强度及持久性，黏合部分具有一定的弹性和韧性；⑦在使用过程中对人体组织无刺激性；⑧具有良好的使用状态并易于保存。
碳酸锌：白色细微无定形粉末。无臭、无味。相对密度4.42-4.45。不溶于水和醇。微溶于氨。能溶于稀酸和氢氧化钠中。
赖氨酸：赖氨酸是人体必需氨基酸之一，能促进人体发育、增强免疫功能，并有提高中枢神经组织功能的作用。赖氨酸为碱性必需氨基酸。作用：1．提高智力、促进生长、增强体质。2．增进食欲、改善营养不良状况。3．改善失眠，提高记忆力。4．帮助产生抗体、激素和酶，提高免疫力、增加血色素。5．帮助钙的吸收，治疗防止骨质疏松症6．降低血中甘油三酯的水平，预防心脑血管疾病的产生。
十大功劳根：基源：为小檗科十大功劳属植物阔叶十大功劳、细叶十大功劳的根。成分：1。阔叶十大功劳根含生物碱、小檗碱。2。华南十大功劳根含生物碱、异粉防己碱、小檗碱、掌叶防己碱、药根碱及小檗胺。药性：《四川中药志》1960年版：“性寒，味苦。”功用主治：清热燥湿，解毒消肿。主治湿热痢疾，腹泻，黄疸，肺痨咳血，咽喉痛，牙痛，目赤肿痛，跌打损伤，疮疡，湿疹，烫伤等。宜忌：脾胃虚寒者慎服。
荆芥穗：基源：本品为唇形科植物荆芥的干燥花穗。性味：辛，微温。归经：归肺、肝经。功效：解表散风，透疹。主治：解表散风，透疹。用于感冒，头痛，麻疹，风疹，疮疡初起。炒炭治便血，崩漏，产后血晕。
桉叶油：无色或微黄色液体。呈特有清凉尖刺桉叶香气并带几分樟脑气味，带些药气，有辣口清凉感，香气强烈而不持久。有一定防霉及杀菌防腐作用。几乎不溶于水，溶于乙醇、无水乙醇、油和脂肪中。用途：桉叶油具有杀菌作用，大量用于医药制品，也可作止咳糖浆、胶姆糖、含漱剂、牙膏、空气清净剂等的赋香剂。
儿茶：基源：本品为豆科植物儿茶的去由枝、干的干燥煎膏。冬季采收技、干，除去外皮，砍成大块，加水煎煮，浓缩，干燥。性状：本品呈方形或不规则块状，大小不一。表面棕褐色或黑褐色，光滑而稍有光泽。质硬，易碎，断面不整齐，具光泽．有细孔，遇潮有蚀性。性味：苦、涩，微寒。归经：归肺经。功效：收湿生肌敛疮。主治：用于溃疡不敛，湿疹，口疮，跌扑伤痛．外伤出血。药理作用：1。保肝、利胆作用；2。抗病原微生物作用；3。抑制肠道运动及抗腹泻作用；4。降血糖作用。
细辛：基源：本品为马兜铃科植物北细辛、汉城细辛或华细辛的根及根茎。化学成份：辽细辛含挥发油约3％，挥发油的主要成分是甲基丁香油酚，其他有黄樟醚、β-蒎烯、优葛缕酮、酚性物质等。性味：辛，温。归经：归心、肺、肾经。药理作用：①局部麻醉作用，②解热、镇痛作用，③抑菌作用。功效：祛风散寒，通窍止痛，温肺化饮。主治：用于风寒感冒，头痛，牙痛。鼻塞鼻渊，风湿痹痛，痰饮喘咳。
防风：基源：本品为伞形科植物防风的干燥根。性状：本品呈长圆锥形或长圆柱形，下部渐细，有的略弯曲。面灰棕色，粗糙、有纵皱纹、多数横长皮孔样突起及点状的细根痕。根头部有明显密集的环纹，有的环纹上残存棕褐色毛状叶基。化学成份：防风含挥发油、甘露醇、苦味甙等。性味：辛、甘．温。归经：归膀胱、肝、脾经。功效：解表祛风，胜湿，止痉。主治：用于感冒头痛．风湿痹痛，风疹瘙痒，破伤风。
本发明提供的一种纳米银抗炎杀菌温敏水凝胶组方科学，制备方法简单。除此之外，本发明的优良效果还表现在：
1、本发明以固相法制成壳聚糖纳米银复合物，并加载多味中药原料，制备具有抗炎杀菌功效的纳米银温敏水凝胶，在室温条件下为可流动胶体溶液，温度升高到体温时可固化形成凝胶, 从而可以缓释加载药物的效果；
2、本发明温敏水凝胶不仅具备纳米银抗菌性，还具备壳聚糖促进细胞生长，减少疤痕生成的作用；能快速有效的促进创面愈合、止血、止痛、抗炎杀菌；
3、本发明温敏水凝胶具有较好的抑菌作用，抗菌谱广、无细胞毒性，高效低毒；均匀喷洒后可在创伤表面形成一层凝胶薄膜，抑菌作用持久、透气性和吸湿性良好。
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，但保护范围不被此限制。
实施例1 一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成（45g/份）：壳聚糖124份；硝酸银0.06份；甘油磷酸纳7份；丝素蛋白42份；丙烯酸酯28，碳酸锌17份；赖氨酸8份；十大功劳根12份；荆芥穗9份；桉叶油12份；儿茶7份；细辛5份；防风6份。
一种纳米银抗炎杀菌温敏水凝胶的制备方法是：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖15分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡1小时，然后文火加热煎煮两次，每次各煎煮26分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨1小时，紫外光照射4小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.05mol/L的醋酸水溶液溶解制成质量浓度为1.5%的壳聚糖纳米银复合物溶液，向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌20分钟，然后经pH调节剂磷酸氢二钠调节pH至6.2；
（5）将（4）所得的溶液放入35℃恒温水浴锅中，6分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
实施例2 一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成（25g/份）：壳聚糖140份；硝酸银0.08份；甘油磷酸纳11份；丝素蛋白60份；丙烯酸酯36，碳酸锌21份；赖氨酸16份；十大功劳根20份；荆芥穗13份；桉叶油18份；儿茶13份；细辛12份；防风9份。
一种纳米银抗炎杀菌温敏水凝胶的制备方法是：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖35分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡2小时，然后文火加热煎煮两次，每次各煎煮44分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨3小时，紫外光照射8小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.15mol/L的醋酸水溶液溶解制成质量浓度为2.5%的壳聚糖纳米银复合物溶液，向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌50分钟，然后经pH调节剂磷酸氢二钠调节pH至7.0；
（5）将（4）所得的溶液放入38℃恒温水浴锅中，3分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
实施例3 一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成（40g/份）：壳聚糖130份；硝酸银0.065份；甘油磷酸纳8份；丝素蛋白48份；丙烯酸酯30，碳酸锌18份；赖氨酸10份；十大功劳根14份；荆芥穗10份；桉叶油14份；儿茶9份；细辛7份；防风7份。
一种纳米银抗炎杀菌温敏水凝胶的制备方法是：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖20分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡1.3小时，然后文火加热煎煮两次，每次各煎煮30分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨1.5小时，紫外光照射5小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.08mol/L的醋酸水溶液溶解制成质量浓度为1.7%的壳聚糖纳米银复合物溶液，向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌30分钟，然后经pH调节剂磷酸氢二钠调节pH至6.4；
（5）将（4）所得的溶液放入36℃恒温水浴锅中，5分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
实施例4 一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成（30g/份）：壳聚糖134份；硝酸银0.075份；甘油磷酸纳10份；丝素蛋白54份；丙烯酸酯34，碳酸锌20份；赖氨酸14份；十大功劳根18份；荆芥穗12份；桉叶油16份；儿茶11份；细辛10份；防风8份。
一种纳米银抗炎杀菌温敏水凝胶的制备方法是：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖30分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡1.7小时，然后文火加热煎煮两次，每次各煎煮40分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨2.5小时，紫外光照射7小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.12mol/L的醋酸水溶液溶解制成质量浓度为2.3%的壳聚糖纳米银复合物溶液，向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌40分钟，然后经pH调节剂磷酸氢二钠调节pH至6.8；
（5）将（4）所得的溶液放入37℃恒温水浴锅中，4分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
实施例5 一种纳米银抗炎杀菌温敏水凝胶，取下列重量份的原料制备而成（35g/份）：壳聚糖132份；硝酸银0.07份；甘油磷酸纳9份；丝素蛋白50份；丙烯酸酯32，碳酸锌19份；赖氨酸12份；十大功劳根16份；荆芥穗11份；桉叶油15份；儿茶10份；细辛8.5份；防风7.5份。
一种纳米银抗炎杀菌温敏水凝胶的制备方法是：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖25分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡1.5小时，然后文火加热煎煮两次，每次各煎煮35分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨2小时，紫外光照射6小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.1mol/L的醋酸水溶液溶解制成质量浓度为2%的壳聚糖纳米银复合物溶液，向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌35分钟，然后经pH调节剂磷酸氢二钠调节pH至6.6；
（5）将（4）所得的溶液放入36.5℃恒温水浴锅中，4.5分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
试验例1 本发明一种纳米银抗炎杀菌温敏水凝胶的抑菌作用试验
1 试验用材料
测试样品：A样品：0.05%含纳米银的温敏水凝胶；B样品：0.025%含纳米银的温敏水凝胶；C样品：0.01%含纳米银的温敏水凝胶；D样品：壳聚糖温敏水凝胶。
培养基：牛肉膏蛋白胨培养基、沙氏培养基、血清培养基。
供试菌株：（1）细菌：革兰氏染色阳性菌（G+）：金黄色葡萄球菌临床菌（聊城市中医医院提供）、金黄色葡萄球菌标准菌株ATCC25923（聊城市人民医院提供）。
革兰氏染色阴性菌（Ｇ-）：大肠杆菌临床菌株、肺炎克雷伯菌临床菌株、阴沟肠杆菌临床菌株、肺炎链球菌临床菌株、铜绿假单胞菌临床菌株（均由聊城市中医医院提供）；大肠杆菌标准菌株ATCC25922、铜绿假单胞菌标准菌株ATCC27853、流感嗜血杆菌临床菌株（均由聊城市人民医院提供）。
（2）真菌：白色念珠菌（由聊城市中医医院提供）、红色毛癣菌（购置于上海北诺生物科技有限公司）、絮状表皮癣菌（购置于上海丰寿生物科技有限公司）。
2.实验方法
2.1各样品对10种细菌的抑菌作用
将保存的各菌种分别进行活化培养，长出单菌落后，用接种环挑取少许菌体于装有9ml无菌生理盐水的试管内，振荡均匀，分别制备107 cfu/ml的悬浮液。在无菌条件下，在90mm的培养皿中加适量的培养基，凝固后加100微升菌液，涂布均匀，静置20-30min。将制备的温敏水凝胶样品分别做成直径为0.4cm的圆形，放置在固体培养基上，同一培养皿上依次放置壳聚糖温敏、0.01%含纳米银的温敏水凝胶、0.025%含纳米银的温敏水凝胶、0.05%含纳米银的温敏水凝胶，重复6次，在37℃的恒温培养箱中培养24h；观察样品与培养基的接触面有无细菌生长，并测量抑菌圈的大小，采用SPSS v11.5软件统计分析，数据以均数±标准差（x±S）表示。
2.2 各样品对3种真菌的抑菌作用
将保存的各菌种分别进行活化培养，分别制备浓度为1×106cfu/ml的菌液,其它操作同上，37℃的恒温培养箱中培养48h，观察样品与培养基的接触面有无细菌生长，测量抑菌圈的大小。采用SPSS软件统计分析，计量资料以均数±标准差（x±S）表示。
3 结果与分析
3.1含纳米银温敏水凝胶对10种细菌：大肠杆菌临床菌株及其标准菌株ATCC25922、金黄色葡萄球菌临床菌株及其标准菌株ATCC25923、铜绿假单胞菌临床菌株及其标准菌株ATCC27853、阴沟肠杆菌、肺炎链球菌、流感嗜血杆菌、产酸克雷伯菌抑菌圈直径见表1。
表1 各样品对10种细菌的接触抑菌抑菌圈直径（单位：cm；样品直径0.4cm）
由表1数据可知，含纳米银的温敏水凝胶及壳聚糖温敏水凝胶对实验所用
10种细菌均具有较好的接触抑菌作用，样品与培养基的接触面均无细菌生长。随着纳米银含量的增加，抑菌效果增强，说明纳米银增强了壳聚糖温敏水凝胶的抑菌作用。
3.2 各样品对三种真菌：白色念珠菌、红色毛癣菌、絮状表皮癣菌的接触抑菌抑菌圈直径见表2。
表2 各样品对三种真菌的接触抑菌抑菌圈直径（单位：cm；样品直径0.4cm）
含纳米银的温敏水凝胶及壳聚糖温敏水凝胶对实验所用三种真菌均具有较好的接触抑菌作用，凝胶样品与培养基的接触面均无真菌生长，随着纳米银含量的增加，抑菌作用增强。含纳米银的温敏水凝胶对絮状表皮癣的的抑菌作用最明显，其次是红色毛癣菌，对白色念珠菌的抑菌作用明显弱于前两种真菌。
4. 结果与讨论
实验结果显示：含纳米银的温敏水凝胶对以大肠杆菌为代表的革兰氏阴性菌，对以金黄色葡萄球菌为代表的革兰氏阳性菌，对以白色念珠菌为代表的真菌，均具有较好的接触抑菌作用。含纳米银温敏水凝胶的抑菌作用优于壳聚糖温敏水凝胶的抑菌作用；对细菌的抑菌作用优于对真菌的抑菌作用；随着纳米银含量的增加，抑菌作用增强，纳米银增强了壳聚糖温敏水凝胶的抑菌作用。
试验例2：本发明所得一种纳米银抗炎杀菌温敏水凝胶（实施例5）的细胞相容性试验
1 样品制备
取本发明所得温敏水凝胶，按照水凝胶样品:细胞培养液=0.2 g:1 mL的比例浸泡，37℃浸泡48 h得浸提液，滤膜过滤除菌，DMEM培养液依次稀释，获得稀释5倍、10倍、20倍、50倍、100倍的水凝胶浸提液。
2、试验方法
通过MTT法测定不同稀释倍数的水凝胶浸提液对NIH-3T3细胞的毒性。
方法：于培养皿中培养NIH-3T3细胞长至80%的状态，弃掉旧的培养液，用37℃的PBS溶液冲洗2两遍，加入1ml 0.25%的胰酶，轻轻晃动，把培养皿置于倒置显微镜下观察细胞的形态，等大部分细胞趋于变圆时，移至洁净工作台上，吸出胰酶，加入2ml 37℃的DMEM细胞培养液终止消化，制成细胞悬液。按1:4比例传入新培养皿中，在37℃，5%CO2 的饱和湿度的培养箱中培养。将对数生长期的细胞按4000个/孔种植到96孔板，在37℃，5% CO2的细胞培养箱中培养，细胞长到80%以上，用浸提48h不同稀释倍数的浸提液处理后，弃去细胞培养液，固定1 h，用SRB染色10 min，然后用乙酸冲洗，晾干，每孔加入100 微升Tris碱溶解，在492nm处测吸光值。细胞存活率（%）=（处理组OD值/对照组OD值）×100%。结果如表3所示。
表3 水凝胶浸提液48h毒性测试结果
毒性的判定标准：0级和I级为合格，Ⅱ级结合形态分析综合评价，Ⅲ～Ⅳ级为不合格。由表3数据可以看出不同浓度的水凝胶浸提液对NIH-3T3细胞RGR均在75％以上，毒性等级为0和I级，表明所有样品浸提液没有明显细胞毒性，具有良好的细胞相容性。
最后应说明的是，实施例只是本发明最优的具体实施方式而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种纳米银抗炎杀菌温敏水凝胶，其特征在于，取下列重量份的原料制备而成：壳聚糖124-140；硝酸银0.06-0.08；甘油磷酸纳7-11；丝素蛋白42-60；丙烯酸酯28-36，碳酸锌17-21；赖氨酸8-16；十大功劳根12-20；荆芥穗9-13；桉叶油12-18；儿茶7-13；细辛5-12；防风6-9。
2.如权利要求1所述的一种纳米银抗炎杀菌温敏水凝胶，其特征在于，取下列重量份的原料制备而成：壳聚糖130-134；硝酸银0.065-0.075；甘油磷酸纳8-10；丝素蛋白48-54；丙烯酸酯30-34，碳酸锌18-20；赖氨酸10-14；十大功劳根14-18；荆芥穗10-12；桉叶油14-16；儿茶9-11；细辛7-10；防风7-8。
3.如权利要求1所述的一种纳米银抗炎杀菌温敏水凝胶，其特征在于，取下列重量份的原料制备而成：壳聚糖132；硝酸银0.07；甘油磷酸纳9；丝素蛋白50；丙烯酸酯32，碳酸锌19；赖氨酸12；十大功劳根16；荆芥穗11；桉叶油15；儿茶10；细辛8.5；防风7.5。
4.如权利要求1-3任一所述的一种纳米银抗炎杀菌温敏水凝胶的制备方法，其特征在于，步骤如下：
（1）将十大功劳根加质量分数为5%的黄酒和质量分数为10%的姜汁拌匀，密闭，隔水炖15-35分钟，至颜色变深，取出，与荆芥穗、儿茶、细辛和防风一同置于煎锅内，加冷水没过药面，浸泡1-2小时（优选浸泡1.3-1.7小时），然后文火加热煎煮两次，每次各煎煮26-44分钟，过滤，滤液减压浓缩至原体积的1/5，加入桉叶油，混匀，得中药浓缩液，备用；
（2）将壳聚糖、硝酸银和谷氨酸混合，充分研磨1-3小时（优选充分研磨1.5-2.5小时），紫外光照射4-8小时，然后加入碳酸锌混匀，得壳聚糖纳米银复合物，备用；
（3）将步骤（2）所得壳聚糖纳米银复合物用0.05-0.15mol/L的醋酸水溶液溶解制成质量浓度为1.5%-2.5%的壳聚糖纳米银复合物溶液（优选制成质量浓度为1.7%-2.3%的壳聚糖纳米银复合物溶液），向该溶液中加入步骤（1）所得中药浓缩液，混匀，得混合溶液，备用；
（4）向步骤（3）所得混合溶液中加入甘油磷酸钠、丙烯酸酯和丝素蛋白，于室温下机械搅拌20-50分钟（优选于室温下机械搅拌30-40分钟），然后经pH调节剂调节pH至6.2-7.0；
（5）将（4）所得的溶液放入35-38℃恒温水浴锅中（优选放入36-37℃恒温水浴锅中），3-6分钟后即发生相转变，由可流动的胶体溶液转变为不流动的水凝胶。
5.根据权利要求4所述的温敏水凝胶的制备方法，其特征在于，步骤（1）隔水炖20-30分钟（优选隔水炖25分钟）。
6.根据权利要求4所述的温敏水凝胶的制备方法，其特征在于，步骤（1）每次各煎煮30-40分钟（优选每次各煎煮35分钟）。
7.根据权利要求4所述的温敏水凝胶的制备方法，其特征在于，步骤（2）紫外光照射5-7小时（优选紫外光照射6小时）。
8.根据权利要求4所述的温敏水凝胶的制备方法，其特征在于，步骤（3）用0.08-0.12mol/L的醋酸水溶液溶解（优选用0.1mol/L的醋酸水溶液溶解）。
9.根据权利要求4所述的温敏水凝胶的制备方法，其特征在于，步骤（4）所述pH调节剂为磷酸氢二钠。
10.根据权利要求4所述的温敏水凝胶的制备方法，其特征在于，步骤（4）调节pH至6.4-6.8（优选调节pH至6.6）。
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