CN110368335A - 乳香提取物在口腔抑菌组合物中的应用 - Google Patents
乳香提取物在口腔抑菌组合物中的应用 Download PDFInfo
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- CN110368335A CN110368335A CN201910553655.4A CN201910553655A CN110368335A CN 110368335 A CN110368335 A CN 110368335A CN 201910553655 A CN201910553655 A CN 201910553655A CN 110368335 A CN110368335 A CN 110368335A
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- olibanum
- oral cavity
- bacteriostasis rate
- olibanum extract
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Abstract
本发明涉及乳香提取物在口腔抑菌组合物中的应用,尤其是在口腔抑菌组合物中的应用,包括在牙膏中的应用,在漱口水中的应用,在泡腾片中的应用,在口香糖中的应用,在糖果中的应用。其对幽门螺杆菌、大肠埃希菌、变形链球菌、牙龈卟啉单胞菌、金黄色葡萄球菌和中间普氏菌均有较强的抑制作用。
Description
技术领域
本发明涉及乳香提取物的新应用,特别涉及乳香提取物在细菌抑制中的应用,确切地说是一种乳香提取物在口腔抑菌组合物中的应用。
背景技术
研究报道乳香具有抗肿瘤功效,同时乳香还被证明用于人脸时能改善皮肤质量和弹性,因此乳香在护肤品和化妆品中的应用较广,而含有乳香提取物的口腔抑菌组合物,能对包括幽门螺杆菌在内的多种细菌均具有较强的抑菌作用未见报道。
由于口腔位于呼吸道、消化道的上游,口腔感染后极易导致咽喉炎、扁桃体炎、气管炎、肺炎、胃溃疡、肠结核等疾病。有研究表明:胃炎、胃溃疡的“元凶”幽门螺旋杆菌不仅可以在胃幽门部检出,也能在口腔、牙、唾液中查出。
另外,据资料统计,我国百分之八十以上的成年人都存在牙齿健康问题,牙龈炎、龋齿、牙周炎等牙齿健康问题屡见不鲜,而人们对牙齿健康问题的重视远没有身体其他部分来的多,殊不知,牙齿健康问题若是一味任其发展也能带来极大的危害,轻则导致牙齿的脱落,影响个人形象和进食问题,重则会引发各种全身性的疾病,对人们的健康造成危害,而牙齿健康问题的起点都源于各种各样的细菌。本研究发现乳香提取物,对包括幽门螺杆菌在内的多种细菌均具有较强的抑菌作用,应用于口腔组合物中可有效祛除和抑制口腔中的各种细菌,进而对预防胃部疾病和口腔健康起到一个很好的预防作用。
发明内容
本发明旨在研究乳香提取物的抑菌作用并应用于口腔用品。所要解决的技术问题是确定乳香提取物对幽门螺杆菌、变形链球菌、大肠埃希菌、牙龈卟啉单胞菌、金黄色葡萄球菌和中间普氏菌的抑制作用并将其制备成口腔抑菌组合物(包含牙膏、漱口水、泡腾片、口香糖、糖果、口腔喷雾剂等)。
本发明采用的技术方案是:
乳香提取物在口腔抑菌组合物中的添加量为组合物总重量的0.0001-15%。
具体抑菌效果为:6h和12h,幽门螺杆菌的抑菌率分别为:95.76%和96.44%;6h和12h,变形链球菌的抑菌率分别为:99.54%和98.15%;6h和12h,大肠埃希菌的抑菌率分别为:99.89%和96.95%;6h和12h,牙龈卟啉单胞菌的抑菌率分别为:98.77%和94.46%;6h和12h,金黄色葡萄球菌的抑菌率分别为:98.43%和95.26%;6h和12h,中间普氏菌的抑菌率分别为:96.73%和96.12%。
附图说明
图1为菌斑生物膜荧光染色结果图。
具体实施方式
实施例1-5为口腔抑菌组合物的具体成分。
实施例1:口腔抑菌牙膏
配方:山梨糖醇 15-45%
水合硅石 5-40%
纯净水 TO100%
聚乙二醇-400 0.1-5%
月桂醇硫酸酯钠 0.1-6%
乳香提取物 0.0001-15%
植酸钠 0.1-5%
香精 0.1-5%
羟甲基纤维素钠 0.1-3%
羟丙基瓜尔胶 0.1-6%
糖精钠 0.1-0.5%
柠檬酸锌 0.01-2%
苯甲酸钠 0.1-3%
Cl77891 0.01-5%
制作方法:先将山梨糖醇加入到真空制膏机中,再加入月桂醇硫酸酯钠充分混合均匀,然后加入大部分水,再次混匀,在真空搅拌状态下,依次加入用少部分水进行分散溶解的乳香提取物、糖精钠、水合硅石、聚乙二醇-400、植酸钠、羟甲基纤维素钠、羟丙基瓜尔胶、柠檬酸锌、苯甲酸钠和Cl77891,在真空状态下,进行高速搅拌充分混匀,最后加入香精,再次在真空状态下,进行高速搅拌充分乳化混匀,即得口腔抑菌牙膏。
实施例2:口腔抑菌漱口水
配方:木糖醇 0.1-6%
山梨醇 0.1-8%
水 TO100%
乙二胺四乙酸二钠 0.01-2%
羟苯甲酯 0.01-2%
乳香提取物 0.0001-15%
乙醇 0.1-3%
薄荷脑 0.01-5%
食用薄荷香精 0.02-3%
蛋白酶葡萄糖内酯 0.01-8%
制作方法:将木糖醇、山梨醇、乙二胺四乙酸二钠、羟苯甲酯、乳香提取物、乙醇、薄荷脑、食用薄荷香精和蛋白酶葡萄糖内酯混合均匀,而后加入水进一步搅拌得到澄清液体,即可得到口腔抑菌漱口水。
实施例3:糖果
配方:琼脂 0.1-5%
柠檬酸钠 10-50%
蔗糖 0.01-5%
淀粉糖浆 5-40%
食用色素/香料/酸 0.5-3%
乳香提取物 0.0001-15%
其余为水。
制作方法:将柠檬酸钠、蔗糖、淀粉糖浆、食用色素/香料/酸和乳香提取物混合均匀,而后加入水进行搅拌,搅拌均匀后加入琼脂即可。
实施例4:泡腾片
配方:乳香提取物 0.0001-15%
碳酸氢钠 20-50 %
酒石酸 10-50 %
蔗糖 1-20%
阿斯巴甜 0.01-5 %
羟丙基纤维素 0.01-2%
聚乙二醇6000 0.05-5 %
食用香精 0.05-0.5%
人工色素 0.05-0.5%,
苯甲酸或苯甲酸钠 0.01-0.1%
甘露醇 TO100%
水、乙醇 适量
制作方法:将碳酸氢钠用熔融的聚乙二醇包裹,粉碎成细粉,然后与乳香提取物、酸剂、甜味剂、崩解剂、矫味剂、香精、着色剂、防腐剂和填充剂混合均匀,制粒,干燥,压片。优选酸、 碱比例, 采用聚乙二醇包裹技术,有效地隔离了酸源, 有利于制剂的稳定性, 解决了泡腾剂放置失效的问题,质量稳定。
根据漱口水的使用量及浓度方面的实际情况,制备的泡腾片,重量在0.4~0.8g之间,建议每次使用25ml,一次一片,持续漱口30秒后吐出。
实施例5:口香糖
配方:胶基 20-40
乳香提取物 0.0001-15%
木糖醇 10-30%
麦芽糖醇 5-20%
山梨醇 1-10%
***树胶 1-10%
食用香精 0.05-0.5%
D-甘露糖醇 5-20%
磷脂(大豆磷脂) 0.05-5%
阿斯巴甜 0.01-5 %
二氧化钛 1-5 %
柠檬酸 0.2-0.8 %
苹果酸 0.2-0.8
水、乙醇 适量
制作方法:全部原料混合均匀后,投入到预热到50℃强力搅拌机中捏合,挤压出条,进入5℃的冷却隧道冷却,在温度为15℃湿度为45-50%的环境下压条整条,并通过高速自动切条包装机制成成品。
下面通过试验对本发明所述的含乳香提取物的口腔抑菌组合物的抑菌效果进行了验证,试验中所用的乳香提取物可自行精炼制备得到,也可直接从市面上进行购置,来源广泛、价格适中;
试验目的:验证口腔抑菌组合物对幽门螺杆菌、口腔常见菌(大肠埃希菌)、牙周致病菌(牙龈卟啉单胞菌)、龋致病菌(变形链球菌)、金黄色葡萄球菌及中间普氏菌的抗菌性能。
1.实验材料与方法
1.1材料
1.1.1实验样本
口腔抑菌组合物(取实施例1-5中的组合物),乳香提取物(乳香为:乳香树Boswellia carterii Birdw及同属植物Boswellia bhaurdajiana Birdw和齿叶乳香Boswelliaserrata)。
1.1.2试剂与仪器
厌氧培养箱(美国GeneScience ,E500),细菌恒温培养箱(上海昕仪,XY-DR-360),生物安全柜(上海利康,HFsafe-1200A2),全波长酶标仪(瑞士TECAN,Infinite 200 Pro),荧光显微镜(奥林巴斯,BX53),可调式移液枪(梅特勒,XLS+),电子天平(德国赛多利斯,BT25S);牛心脑肉汤培养基(北京华越洋,GU9-2814),布氏肉汤培养基(北京索莱宝,LA3560),LB培养基(北京索莱宝,L1010),MH培养基(Oxoid公司),哥伦比亚血琼脂培养基(江门凯林贸易有限公司),氯化血红素(北京索莱宝,H8130),WST-8试剂盒(日本同仁,M439),微生物活性荧光染色试剂盒(美国赛默,L7012)。
1.1.3供试病原菌菌株
幽门螺杆菌(Helicobacter pylori HP)、大肠埃希菌(Escherichia coli EC)、变形链球菌(Streptococcus mutans SM)、牙龈卟啉单胞菌(Porphyromonas gingivalis PG)、金黄色葡萄球菌(Staphylococcus aureus SA)、中间普氏菌(Prevotella intermedia PI)
1.2实验方法:
1.2.1培养基的配置:
配置常规布氏培养基用于幽门螺杆菌的培养:常规布氏培养基5g溶于100mL蒸馏水中高压蒸汽121℃灭菌25分钟,冷却时加入1mL抗生素溶液(包含1μg万古霉素,250 IU多粘菌素,0.2mg两性霉素),加入7mL的兔血备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。配置BHI培养基用于变形链球菌的培养:牛心脑浸液培养基粉末,加蒸馏水配成3.7%浓度,高压蒸汽121℃灭菌25分钟后,置于超净台内冷却,备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。
配置BHI血琼脂培养基用于牙龈卟啉单胞菌的培养:牛心脑浸液培养基,加蒸馏水配成3.7%浓度,高压蒸汽121℃灭菌25分钟后加入氯化血红素,使其终浓度为5μg/mL,备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。
配置MH培养基用于金黄色葡萄球菌的培养:称取培养基粉末21.0g,加热溶解于1000ml 蒸馏水中,121℃ 高压灭菌 25分钟后,置于超净台内冷却,备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。
配置哥伦比亚培养基用于中间普氏菌的培养:称取培养基粉末39克,加热溶解于1000ml蒸馏水中,分装,121℃高压灭菌25分钟,冷至45-50℃左右时,加入相当于20mg庆大霉素的无菌硫酸庆大霉素,混匀备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。
配置LB培养基用于大肠埃希菌的培养:LB培养基粉末加蒸馏水配成2.5%浓度,高压蒸汽121℃灭菌25分钟后,置于超净台内冷却,备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。
配置常规布氏培养基用于中间普氏菌的培养:常规布氏培养基5g溶于100mL蒸馏水中高压蒸汽121℃灭菌25分钟,冷却时加入1mL抗生素溶液(包含1μg万古霉素,250 IU多粘菌素,0.2mg两性霉素),加入7mL的兔血备用。对应琼脂板制备加入1.5%琼脂,冷却至50℃倒板,备用。
1.2.2菌株复苏与培养
幽门螺杆菌复苏:将幽门螺杆菌冻干菌株接种于布氏血琼脂平板上,在37℃厌氧(体积比:85% N2,5% H2,10% CO2)培养48小时。
幽门螺杆菌培养:挑已复苏菌株接种于布氏培养基中置于37℃厌氧培养箱中,用真空泵将培养罐内气压抽真空后充入不含氧气的混合气(5% H2,10% C02,85 %N2)。培养48小时后,采用麦氏比浊法用PBS,配成细菌混悬液(108CFU/mL),备用。
大肠埃希菌复苏:将大肠埃希菌冻干菌株接种于LB琼脂平板上,在37℃细菌恒温培养箱中(含5% CO2)培养24小时。
大肠埃希菌培养:挑已复苏的大肠埃希菌菌株接种于LB液体培养基中培养24小时,采用麦氏比浊法用PBS,配成细菌混悬液(108CFU/mL),备用。
变形链球菌复苏:将变形链球菌冻干菌株接种于BHI琼脂平板上,在37℃细菌恒温培养箱中(含5% CO2)培养24小时。
变形链球菌培养:挑已复苏的变形链球菌菌株接种于BHI液体培养基中培养24小时,采用麦氏比浊法用PBS,配成细菌混悬液(108CFU/mL),备用。
牙龈卟啉单胞菌复苏: 将牙龈卟啉单胞菌冻干菌株接种于BHI血琼脂平板上,在37℃厌氧(体积比:80% N2,10% H2,10% CO2)培养48小时。
牙龈卟啉单胞菌培养:挑已复苏的牙龈卟啉单胞菌菌株接种于含血BHI液体培养基中厌氧培养48小时,采用麦氏比浊法用PBS配成细菌混悬液(108CFU/mL),备用。
金黄色葡萄球菌复苏:将金黄色葡萄球菌冻干菌株接种于MH琼脂平板上,在37℃细菌恒温培养箱中(含5% CO2)培养24小时。
金黄色葡萄球菌培养:挑已复苏的金黄色葡萄球菌菌株接种于MH液体培养基中培养24小时,采用麦氏比浊法用PBS,配成细菌混悬液(108CFU/mL),备用。
中间普氏菌复苏:将中间普氏菌冻干菌株接种于哥伦比亚血琼脂平板上,在37℃厌氧(体积比:85% N2,5% H2,10% CO2)培养48小时。
中间普氏菌培养:挑已复苏菌株接种于哥伦比亚血琼脂培养基中置于37℃厌氧培养箱中,用真空泵将培养罐内气压抽真空后充入不含氧气的混合气(5% H2,10% C02,85 %N2)。培养48小时后,采用麦氏比浊法用PBS,配成细菌混悬液(108CFU/mL),备用。
1.2.3实验分组
精确称量0.28g乳香提取物,超声分散于100mL的6种待测细菌对应的培养基,取2mL该溶液,加入2μL待测细菌混悬液,记为乳香提取物实验组(药物终浓度为:0.0028g/ml乳香提取物);精确称量相当于0.28g乳香提取物的口腔抑菌组合物,超声分散于100mL的6种待测细菌对应的培养基,取2mL该溶液,加入2μL待测细菌混悬液,记为口腔抑菌组合物实验组(药物终浓度为:0.0028g/ml乳香提取物);取2mL待测细菌对应的培养基,加入2μL待测细菌混悬液,记为阳性对照;取2mL待测细菌对应的培养基作为阴性对照。实验分组如表1。实验在24孔板内进行,每组设置3个平行样本。
表1为口腔抑菌组合物口腔细菌敏感试验实验分组情况
1.2.4抗菌性能检测:
各实验组分别在对应培养条件下培养6和12小时,采用WST-8试剂盒测试各组样本抗菌性能:各实验组培养既定时间后,各组取3个样本,每个样本0.15mL,加至96孔板,随后96孔板每孔分别加入15μL WST-8试剂,混匀后于37℃下孵育1h后,利用全波长酶标仪测量OD450nm。抗菌率计算公式如下:
抗菌率=(OD阳性对照-OD实验组)/(OD阳性对照-OD阴性对照)×100%
其中,OD阳性对照为阳性对照组待测液的吸光度值,OD实验组为各实验组待测液对应的吸光度值,OD阴性对照为阴性对照组对应的待测液的吸光度值。本实验取抗菌率>95%具有抗菌效果,抗菌率大于80%具有抑菌效果。
为进一步证实抗菌效果,采用L7012染色试剂盒对各分组的表面的菌斑生物膜及死活细菌进行了染色,使用FV3000激光共聚焦显微镜观察细菌的生存状态及菌斑生物膜的附着情况。具体操作如下:取无菌玻片置于无菌24孔板中,加入0.22μm 滤器过滤除菌的人工唾液包被12小时,随后如表1加样,培养12小时后吸弃菌液,以无菌PBS轻洗玻片表面,每孔加入1ml L7012染料后避光冷藏染色15分钟,轻洗后置于荧光显微镜下观察并随机选择视野拍照,其中荧光绿染部分为活菌,荧光红染部分为死菌。
1.2.5统计学方法
使用SPSS 21.0软件进行数据处理,采用单因素方差分析,取P<0.05具有统计学意义。
2.实验结果
2.1口腔抑菌组合物处理及不同处理时间对不同细菌的抑菌效果
2.1.1 WST-8测试各分组抑菌效率
将各组108CFU/mL的菌液使用对应含药培养基稀释1000倍后培养6h及12h。抑菌效果如下:
幽门螺杆菌培养6h后,乳香提取物实验组抑菌率为93.59%、口腔抑菌组合物实验组抑菌率为95.76%。培养12h后,乳香提取物实验组抑菌率为88.75%口腔抑菌组合物实验组抑菌率为96.44%。变形链球菌培养6h后,乳香提取物实验组抑菌率为99.75%、口腔抑菌组合物实验组抑菌率为99.54%。培养12h后,乳香提取物实验组抑菌率为97.67%、口腔抑菌组合物实验组抑菌率为98.15%。
大肠埃希菌培养6h后,乳香提取物实验组抑菌率为99.68%、口腔抑菌组合物实验组抑菌率为99.89%。培养12h后,乳香提取物实验组抑菌率为96.46%、口腔抑菌组合物实验组抑菌率为96.95%。
牙龈卟啉单胞菌培养6h后,乳香提取物实验组抑菌率为94.74%、口腔抑菌组合物实验组抑菌率为98.77%。培养12h后,乳香提取物实验组抑菌率为91.45%、口腔抑菌组合物实验组抑菌率为94.46%。
金黄色葡萄球菌培养6h后,乳香提取物实验组抑菌率为98.97 %、口腔抑菌组合物实验组抑菌率为98.43%。培养12h后,乳香提取物实验组抑菌率为96.73%、口腔抑菌组合物实验组抑菌率为95.26%。
中间普氏菌培养6h后,乳香提取物实验组抑菌率为94.26%、口腔抑菌组合物实验组抑菌率为96.73%。培养12h后,乳香提取物实验组抑菌率为92.46%、口腔抑菌组合物实验组抑菌率为96.12%。
综上数据,乳香提取物实验组、口腔抑菌组合物实验组间见统计学差异(P<0.05),各含有乳香提取物成分的分组表现优异的抗菌效果,而空白对照组口腔用品未见抑菌趋势。口腔抑菌组合物实验组抑菌效果较乳香提取物实验组有更佳的抑菌趋势,口腔抑菌组合物实验组抑菌效果亦优于乳香提取物实验组,但差异无明显统计学意义(P>0.05)。
2.1.2 微生物活性荧光染色试剂盒染色结果
表2为微生物活性荧光染色试剂盒染色结果
如图1所示(荧光绿染部分为活菌,荧光红染部分为死菌),各细菌阳性对照组样本表面见大量绿色荧光,即有大量活菌存在。相同处理条件下,变形链球菌、大肠埃希菌及金黄色葡萄球菌间未见明显差异,牙龈卟啉单胞菌、中间普氏菌和幽门螺杆菌间亦未见明显差异,牙龈卟啉单胞菌、中间普氏菌和幽门螺杆菌对应的阳性对照组表面绿色荧光与变形链球菌、大肠埃希菌对应的阳性对照组相比较少,考虑其原因可能与厌氧菌定植数量较需氧菌少有关。不同细菌对应的乳香提取物实验组与口腔抑菌组合物实验组样本表面见大面积红色荧光与少量绿色荧光,即大量细菌被杀灭。综合上图分析表明,乳香提取物实验组、口腔抑菌组合物实验组成分抑菌效果显著。本部分结果与先前实验结果吻合。
试验结论:
1、实验结果证明口腔抑菌组合物(含乳香提取物),对幽门螺杆菌和牙龈部位相关菌种具有显著的抗菌效果,且与乳香提取物抗菌效果相当,明显优于对照组口腔用品。
2、实验结果证明口腔抑菌组合物(含乳香提取物)能迅速抗菌,且抗菌时间长达12小时。
Claims (9)
1.本发明涉及乳香提取物在口腔抑菌组合物中的应用。
2.本发明涉及乳香提取物在牙膏中的应用。
3.本发明涉及乳香提取物在漱口水中的应用。
4.本发明涉及乳香提取物在泡腾片中的应用。
5.本发明涉及乳香提取物在口香糖中的应用。
6.本发明涉及乳香提取物在糖果中的应用。
7.本发明涉及乳香提取物在口腔喷雾剂中的应用。
8.如权利要求1-7所述的乳香提取物的应用,其特征在于,所述乳香提取物在组合物中的添加量为重量百分比0.0001-15%。
9.如权利要求8所述的乳香提取物的应用,其特征在于:6h和12h,幽门螺杆菌的抑菌率分别为:95.76%和96.44%;6h和12h,变形链球菌的抑菌率分别为:99.54%和98.15%;6h和12h,大肠埃希菌的抑菌率分别为:99.89%和96.95%;6h和12h,牙龈卟啉单胞菌的抑菌率分别为:98.77%和94.46%;6h和12h,金黄色葡萄球菌的抑菌率分别为:98.43%和95.26%;6h和12h,中间普氏菌的抑菌率分别为:96.73%和96.12%。
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