CN107714649B - 一种抑制口腔致病菌的口腔喷雾剂及其应用 - Google Patents
一种抑制口腔致病菌的口腔喷雾剂及其应用 Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
本发明涉及一种抑制口腔致病菌的口腔喷雾剂及其应用，所述的口腔喷雾剂含有以下重量百分比的组分：叶用莴苣多糖5％～10％，碳酸氢钠1％～2％，苯甲酸钠1％‑2.5％，维生素C 1％‑2.5％，薄荷油和柠檬油共5％‑10％，羟丙基甲基纤维素、透明质酸和黄原胶共3％‑6％，余量为水。经细菌涂敷喷涂实验，显示本发明的口腔喷雾剂能够显著抑制变异链球菌和远缘链球菌的活性，表明该口腔喷雾剂能够有效附着牙齿介质，发挥抑制口腔疾病致病菌的作用。
Description
技术领域
本发明涉及防治口腔疾病的药物和保健品，具体地说，涉及一种抑制口腔致病菌的口腔喷雾剂及其应用。
背景技术
口腔疾病是一类发病率很高的疾病总称，常见的有龋齿、口腔异味、牙痛、牙龈出血等。人的口腔中存在大量微生物，有致病菌、非致病菌和条件致病菌，具体种类相当繁杂。常见的细菌有葡萄球菌、链球菌、涎链球菌、奈瑟氏菌、范永氏球菌、棒状杆菌、以色列放线菌、痰螺旋菌、痰弧菌属、色柔氏螺旋体等，最主要的是链球菌、葡萄球菌和乳酸杆菌。口腔中有弱碱性唾液、食物残渣等，为菌群的繁殖提供了合适条件。口腔中致病菌的存在和大量繁殖是导致大多数口腔疾病的重要原因。如厌氧链球菌中有一种变异链球菌，该菌能分解食物中的蔗糖产生高分子量、粘度大的不溶性葡聚糖，以致将口腔中其它菌群粘附于牙齿表面而形成菌斑，最终导致龋齿。
口香糖、口腔喷雾剂及漱口水是目前生活中常见的口腔保健品，能够在一定程度上达到清新口气、预防龋齿的效果。口腔喷雾剂便于携带，使用方便，不会产生废弃物，在空气中形成雾状进入口腔，可以作用到口腔的各个部位，如上腭、咽喉部等处。专利文献CN106511471A，公开了一种无花果口腔喷雾剂及其制备方法，该无花果口腔喷雾剂以1000mL计，由以下组分组成：无花果果实提取物冻干粉50g-150g、防腐剂2g-6g、吸收促进剂40g-80g和余量为去离子水，所述的吸收促进剂由风轮草皂苷和胆酸盐组成，该无花果口腔喷雾剂，具有抑菌、消炎、止血、润喉、脱臭等多种功效。专利文献CN102198065A，公开了一种口腔喷雾剂及其制备方法，采用浓缩提炼的方式，先将罗汉果、金银花、甘草、野菊花、薄荷和淡竹叶采用浓缩提取的方式，制备植物混合提取物，然后再与甜菊、柿子等其他组分混合制备口腔喷雾剂，该发明的口腔喷雾剂为一种全新植物配方，可滋润口腔，令口气清新，减轻口腔异味，无酒精等添加剂，无毒副作用。
叶用莴苣(var.ramosaHort.)，俗称生菜，属菊科莴苣属，为一年生或二年生草本，是日常餐桌常见绿叶菜之一。具有清热安神、清肝利胆和养胃的功效，所含的莴苣素能够阵痛催眠、降低胆固醇和辅助治疗神经衰弱，甘露醇等成分能够利尿和促进血液循环。
目前还未见提取叶用莴苣多糖，并用于制成口腔喷雾剂，有效防治口腔疾病的报道。
发明内容
本发明利用微波提取的方法从叶用莴苣中提取得到叶用莴苣多糖，将其与促进多糖溶解和粘附的组分组配成喷雾剂，进一步验证了该喷雾剂防治口腔致病菌的效果，在此基础上完成了本发明。
本发明提供了一种抑制口腔致病菌的口腔喷雾剂，所述的口腔喷雾剂含有以下重量百分比的组分：叶用莴苣多糖5％～10％，碳酸氢钠1％～2％，苯甲酸钠1％-2.5％，维生素C1％-2.5％，薄荷油和柠檬油共5％-10％，羟丙基甲基纤维素、透明质酸和黄原胶共3％-6％，余量为水；所述的叶用莴苣多糖其提取方法为：
1)将叶用莴苣的叶片充分干燥粉碎，过60-80目筛；
2)向叶用莴苣粉末加入30-60％乙醇回流提取1-2h，料液比为1:5-8，然后除去乙醇，于40-50℃恒温干燥箱中烘干；
3)将烘干后的叶用莴苣粉末加入到0.5-1％的醋酸水溶液中进行微波提取，料液比为1:5-8，功率300-400W，单次提取时间20-30s，提取次数2-3次，间隔5-10min；
4)合并提取液，在40-50℃下旋转蒸发浓缩至原体积的1/4-1/2，静置过滤，向滤液中加入含15-20％硫酸钠的体积分数30-60％的乙醇溶液，于40-50℃下振荡1-2h，静置，收集硫酸钠相；
5)向收集的硫酸钠相中加入2-4倍体积的无水乙醇溶液沉淀多糖，于40-50℃下振荡1-2h，静置12-24h，5000-8000r/min离心5-10min，收集沉淀；
6)再加入无水乙醇洗涤沉淀2次，于40-50℃恒温干燥箱中烘干至恒重。
所述的薄荷油和柠檬油比例为0.8-1.2。
所述的薄荷油和柠檬油比例为1。
所述的羟丙基甲基纤维素、透明质酸和黄原胶的比例为1:3:2。
本发明还提供了所述的口腔喷雾剂的制备方法，包括以下步骤：提取叶用莴苣多糖，与碳酸氢钠、苯甲酸钠、维生素C和透明质酸共同溶解于水中，搅拌，待溶解充分后，加入薄荷油、柠檬油、羟丙基甲基纤维素和黄原胶，水浴加热搅拌，加热过程中补足水，直至所有成分完全溶解，趁热过滤除菌。
所述水浴加热的温度为40-50℃。
所述过滤除菌使用的滤膜孔径为0.22μm。
本发明还提供了所述的口腔喷雾剂在制备防治口腔疾病的药物或保健品中的应用。
本发明还提供了所述的口腔喷雾剂在制备防治口腔致病菌的药物或保健品中的应用。
所述的口腔致病菌是变异链球菌或远缘链球菌。
本发明的有益效果在于：
1、本发明利用微波提取的方法从叶用莴苣中提取得到叶用莴苣多糖，将其作为活性成分制成口腔喷雾剂，经用鸡蛋壳模拟牙齿进行细菌涂敷喷涂实验，显示本发明的口腔喷雾剂能够显著抑制变异链球菌和远缘链球菌的活性，表明该口腔喷雾剂能够有效附着牙齿介质，发挥抑制口腔疾病致病菌的作用。
2、本发明的喷雾剂选用了羟丙基甲基纤维素、透明质酸和黄原胶成分，能够显著提高抑菌成分的附着力。
3、本发明喷雾剂在添加了柠檬油后，显示出抑菌效果的提高，这可能与柠檬油促进了喷雾剂中各成分溶解和在介质表明的附着力有关。
4、本发明喷雾剂的原料组成简单，便于生产制备。
5、本发明喷雾剂的原料均无毒副作用，安全性好。
6、本发明制备的为喷雾剂型口腔药物或保健品，使用方便，不产生废弃物。
具体实施方式
下面通过具体实施例对本发明作进一步的详细说明。
实施例1
1实验材料
叶用莴苣品种前卫75，鸡蛋：购于菜市。
试剂：碳酸氢钠、苯甲酸钠、羟丙基甲基纤维素、透明质酸、黄原胶，购于国药集团化学试剂有限公司；维生素C，购于南京化学试剂股份有限公司；薄荷油、柠檬油，购于上海江莱生物科技有限公司。
设备：DU-640紫外/可见分光光度计：美国贝克曼公司；AUW320分析天平：日本岛津；MA-2270EGC海尔微波炉：青岛海尔微波制品有限公司；FW100型高速粉碎机：北京中兴伟业仪器有限公司；RE52CS旋转蒸发仪：上海亚荣生化仪器厂；DZF真空干燥箱：上海海向仪器设备厂；800型台式离心机：上海手术仪器厂。
菌株：变异链球菌CGMCC12500，购于中国普通微生物菌种保藏管理中心；远缘链球菌ATCC27351，购于北京北纳创联生物技术研究院。
培养基：TPY培养基、BHI培养基，购于青岛海博生物技术有限公司。
2试验方法
2.1口腔喷雾剂的配制
1)取新鲜叶用莴苣的叶片，于45℃恒温干燥箱内充分干燥，粉碎机粉碎，过80目筛；
2)向叶用莴苣粉末加入50％乙醇回流提取1.5h，料液比1:6，然后除去乙醇，于45℃恒温干燥箱中烘干；
3)将烘干后的叶用莴苣粉末加入到0.8％的醋酸水溶液中进行微波提取，料液比1:6，功率350W，单次提取时间25s，提取次数2次，间隔10min；
4)合并提取液，在45℃下旋转蒸发浓缩至原体积的1/2，静置过滤，向滤液中加入含15％硫酸钠的体积分数50％的乙醇溶液，于45℃下振荡1.5h，静置，收集硫酸钠相；
5)向收集的硫酸钠相中加入3倍体积的无水乙醇溶液沉淀多糖，于45℃下振荡1.5h，静置18h，6000r/min离心8min，收集沉淀；
6)再加入无水乙醇洗涤沉淀2次，于45℃恒温干燥箱中烘干至恒重；
7)按照表1中各成分的用量，配制不同组分组成的口腔喷雾剂。制备方法：称取提取叶用莴苣多糖至干净烧杯中，加入碳酸氢钠、苯甲酸钠、维生素C、透明质酸和适量纯水，不断搅拌，至所有成分溶解充分后，加入薄荷油、柠檬油、羟丙基甲基纤维素(或羧甲基纤维素钠)和黄原胶，于45℃水浴锅中加热搅拌，加热过程中补足水至100ml，直至所有成分完全溶解，趁热用0.22μm孔径的滤膜除菌器过滤除菌。
表1口腔喷雾剂的组分组成
2.2口腔喷雾剂的抑菌试验
2.2.1菌悬液制备
变异链球菌接种于TPY培养基，远缘链球菌接种于BHI培养基，厌氧培养18h，挑取琼脂表面的单个菌落混悬于相应液体培养基中培养，至菌液浓度1*105CFU/mL，将菌液等体积混合，即细菌原液。
2.2.2细菌涂布及消毒
用微量移液器吸取2ml菌液，用未脱脂无菌棉签蘸取细菌悬液在经煮沸彻底消毒且无炸裂的鸡蛋表面均匀涂抹直至菌液被涂完，随后不同组别分别使用以上喷雾剂1-8喷雾消毒，喷嘴距鸡蛋表面10cm左右，以每秒1次的频率，将单个鸡蛋分4个方向正面喷涂，单面喷涂2次，控制喷涂压力一致。以不含叶用莴苣多糖制备的喷雾剂作为阴性对照，该阴性对照喷雾剂中其他各组分及含量与喷雾剂1的相同。每组做三个平行实验。
2.2.3细菌取样培养
喷涂10min后取样，每个鸡蛋加注100ml无菌生理盐水，用振荡器以恒定低功率慢速振荡1min，再用微量移液器吸取10μl液体，接种于培养平板，厌氧培养48h后，进行CFU计数，计算抑菌率。抑菌率计算公式为：[(阴性对照组菌落数—处理组菌落数)/阴性对照组菌落数]*100％。
2.2.4统计分析
采用SPSS13.0软件进行单因素方差分析，p<0.05为差异具有统计学意义。
3实验结果
对以上喷雾剂1-8喷涂后10min取样进行CFU计数，统计结果见表2-3。分析结果发现，喷雾剂1-4对变异链球菌和远缘链球菌表现出显著的抑制作用，抑菌率达到70.0％及以上。喷雾剂5-8的抑菌率明显低于喷雾剂1-4的的抑菌率，与喷雾剂1-4的抑菌率两两比较，差异均具有统计学意义(p<0.05)。实验表明喷雾剂中黏附剂的选择对于抑菌效果来说具有显著的影响，同时，添加柠檬油可显著提高抑菌效果。
表2各口腔喷雾剂处理的CFU计数结果
| 组别 | 菌落数(CFU/ml) | 抑菌率(％) |
| 喷雾剂1 | 78±13 | 79.8 |
| 喷雾剂2 | 101±25 | 73.9 |
| 喷雾剂3 | 116±32 | 70.0 |
| 喷雾剂4 | 96±27 | 75.2 |
| 喷雾剂5 | 203±35 | 47.5 |
| 喷雾剂6 | 186±40 | 51.9 |
| 喷雾剂7 | 189±36 | 51.2 |
| 喷雾剂8 | 214±33 | 44.7 |
| 阴性对照 | 387±58 | - |
*变异链球菌。
表3各口腔喷雾剂处理的CFU计数结果
| 组别 | 菌落数(CFU/ml) | 抑菌率(％) |
| 喷雾剂1 | 99±13 | 84.8 |
| 喷雾剂2 | 146±25 | 77.6 |
| 喷雾剂3 | 160±32 | 75.4 |
| 喷雾剂4 | 123±27 | 81.1 |
| 喷雾剂4 | 317±35 | 51.3 |
| 喷雾剂5 | 224±40 | 65.6 |
| 喷雾剂6 | 215±36 | 68.0 |
| 喷雾剂4 | 293±33 | 55.0 |
| 阴性对照 | 651±58 | - |
*远缘链球菌。
Claims (8)
1.一种抑制口腔致病菌的口腔喷雾剂，其特征在于，所述的口腔喷雾剂含有以下重量百分比的组分：叶用莴苣多糖 5%～10%，碳酸氢钠1%～2%，苯甲酸钠1%-2.5%，维生素C 1%-2.5%，薄荷油和柠檬油共5%-10%，羟丙基甲基纤维素、透明质酸和黄原胶共3%-6%，余量为水；所述的叶用莴苣多糖其提取方法为：
1）将叶用莴苣的叶片充分干燥粉碎，过60-80目筛；
2）向叶用莴苣粉末加入30-60%乙醇回流提取1-2h，料液比为1:5-8，然后除去乙醇，于40-50℃恒温干燥箱中烘干；
3）将烘干后的叶用莴苣粉末加入到0.5-1%的醋酸水溶液中进行微波提取，料液比为1:5-8，功率300-400W，单次提取时间20-30s，提取次数2-3次，间隔5-10min；
4）合并提取液，在40-50℃下旋转蒸发浓缩至原体积的1/4-1/2，静置过滤，向滤液中加入含15-20%硫酸钠的体积分数30-60%的乙醇溶液，于40-50℃下振荡1-2h，静置，收集硫酸钠相；
5）向收集的硫酸钠相中加入2-4倍体积的无水乙醇溶液沉淀多糖，于40-50℃下振荡1-2h，静置12-24h，5000-8000r/min离心5-10min，收集沉淀；
6）再加入无水乙醇洗涤沉淀2次，于40-50℃恒温干燥箱中烘干至恒重。
2.根据权利要求1所述的口腔喷雾剂，其特征在于，所述的薄荷油和柠檬油比例为0.8-1.2。
3.根据权利要求2所述的口腔喷雾剂，其特征在于，所述的薄荷油和柠檬油比例为1。
4.根据权利要求1所述的口腔喷雾剂，其特征在于，所述的羟丙基甲基纤维素、透明质酸和黄原胶的比例为1:3:2。
5.权利要求1-4任一权利要求所述的口腔喷雾剂的制备方法，其特征在于，包括以下步骤：提取叶用莴苣多糖，与碳酸氢钠、苯甲酸钠、维生素C和透明质酸共同溶解于水中，搅拌，待溶解充分后，加入薄荷油、柠檬油、羟丙基甲基纤维素和黄原胶，水浴加热搅拌，加热过程中补足水，直至所有成分完全溶解，趁热过滤除菌。
6.根据权利要求5所述的制备方法，其特征在于，所述水浴加热的温度为40-50℃。
7.根据权利要求5所述的制备方法，其特征在于，所述过滤除菌使用的滤膜孔径为0.22μm。
8.权利要求1-4任一权利要求所述的口腔喷雾剂在制备防治口腔致病菌的药物或保健品中的应用，所述的口腔致病菌是变异链球菌或远缘链球菌。
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