CN117482048A - 一种含有天然活性成分的水包油型纳米乳及其制法和应用 - Google Patents
一种含有天然活性成分的水包油型纳米乳及其制法和应用 Download PDFInfo
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Abstract
本发明公开的一种含有天然活性成分的水包油型纳米乳及其制法和应用,水包油型纳米乳包括如下重量百分含量的组分:表面活性剂2%~30%,油相5%~45%,黄芩苷1~2%,人参皂苷1~2%,苦参碱1~2%,泛醇0.01%~1%,赖氨酸0.05%~2%,苏氨酸0.1%~0.3%,锌盐0.05%~0.3%,防腐剂0.1%~1%,余量为水;本发明含有纳米乳包载的天然活性成分,天然温和,增加了产品的稳定性,减少刺激性,涂抹皮肤后,易于吸收,可预防头发脱落,滋养毛囊,扩张血管,为发根增加营养和氧气;强韧固发,增加发量,同时具有抗氧化作用,延缓衰老,滋养皮肤。
Description
技术领域
本发明涉及一种水包油型纳米乳,尤其涉及一种含有天然活性成分的水包油型纳米乳,还涉及其制法和应用。
背景技术
脱发历来是困扰人类的常见而顽固的疾病,毛发不仅对人体有保护作用,而且也是机体健康的标志,脱发产生的心理影响难以估量,严重影响人们的生活。随着人们生活节奏不断加快,生活和工作压力变大,脱发已成为社会普遍存在的问题并呈低龄化趋势。
黄芩苷(baicalin)是黄芩的主要活性成分,黄芩是中医理论中运用治疗脱发和促进毛发生长的重要中药药材,黄芩苷是从黄芩中提取的黄酮类成分,已经有大量文献报道表明其具有抗氧化、抗炎、抗病毒、促进毛发生长等多种作用。黄芩苷和葡萄糖醛酸苷之间可以形成分子内氢键,导致低溶解度,从而大大限制了黄芩苷的临床应用。与黄芩苷相同,多种中药成分都具有溶解性差、渗透性差、活性成分难以充分发挥作用等问题,这些问题极大的限制了天然活性中药成分的应用。
发明内容
发明目的:本发明提供了一种载药量高、包封率高、无刺激性的含有天然活性成分的水包油型纳米乳,还提供上述水包油型纳米乳的制法和应用。
技术方案:本发明公开了一种含有天然活性成分的水包油型纳米乳,包括如下重量百分含量的组分:表面活性剂2%~30%,油相5%~45%,黄芩苷1~2%,人参皂苷1~2%,苦参碱1~2%,泛醇0.01%~1%,赖氨酸0.05%~2%,苏氨酸0.1%~0.3%,锌盐0.05%~0.3%,防腐剂0.1%~1%,余量为去离子水。
优选的,所述纳米乳的乳滴粒径为1~200nm。
优选的,所述油相为肉豆蔻酸异丙酯、肉豆蔻酸肉豆蔻醇酯、棕榈酸异丙酯、亚油酸异丙酯、苯甲酸十二醇酯、异硬脂酸异硬酯醇酯、脂肪酸乳酸酯、油酸癸酯或棕榈酸辛酯。
优选的,所述防腐剂为苯氧乙醇或尼泊金甲酯。
优选的,所述表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂与助表面活性剂的质量比(Km)为1~3:1。
优选的,所述主表面活性剂由质量比为1~3:1的聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚组成,所述主表面活性剂的亲水亲油平衡值为11.8~13.4。
优选的,所述助表面活性剂为无水乙醇。
本发明还公开了水包油型纳米乳的制备方法,包括以下步骤:
(1)取聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚,混合均匀得到主表面活性剂,主表面活性剂和助表面活性剂按照质量比1~3:1混合得到表面活性剂;
(2)将黄芩苷溶解于油相中,加入表面活性剂混合均匀得到混合物A;
(3)将人参皂苷、苦参碱、泛醇、苏氨酸和赖氨酸溶于水中,得到混合物B;
(4)将混合物B加入混合物A中,持续搅拌均匀,得到含有天然活性成分的水包油型纳米乳。
优选的,步骤(1)中的搅拌混合温度为30~37℃。
上述水包油型纳米乳在制备治疗脱发、促进毛发生长的药物或洗护用品中的应用。
发明原理:本发明的含有天然活性成分的水包油型纳米乳,是由油相、水相、表面活性剂和助表面活性剂组成的粒径为1~200nm的均相体系,具有良好的载药性和药物溶解性以及亲水亲油性。
在所选择的中药成分中,苦参碱是苦参中的活性成分,苦参为是豆科植物苦参的干燥根,具有清热祛湿、抗菌消炎等功效。目前,研究表明其含有的苦参碱及氧化苦参碱有免疫抑制调节、抗炎和抗雄性激素作用,随着生活节奏的加快,人们压力增大或焦虑紧张,多伴有头皮油腻、头发瘙痒、头屑增多等,苦参碱同样可起到清热祛湿、抗菌消炎的作用,从而减少头屑产生。
人参性平、味甘、微苦,微温。归脾、肺经、心经。具有“大补元气,补脾益肺,安神益智”之功效。可以有效缓解失眠,并且人参的补气益血的功效可以促进头部血液循环,使气血能够滋养发根,促进毛发的生长。同时,人参具有提升免疫力的作用,也在一定程度上增强其防脱发作用。人参皂苷对头发的角质形成细胞增殖有保护作用,对头发生长有促进作用;苦参碱具有免疫抑制调节、抗炎和抗雄性激素作用;黄芩苷具有抗氧化、抗炎、抗病毒、促进毛发生长等多种作用,协同达到防脱发效果的提升。
通过载入纳米乳制成相应治疗制剂,解决了黄芩苷的BA分子结构中黄酮和葡萄苷酸之间能够形成分子内氢键,导致其水溶性和脂溶性均较差,在水中溶解度低、溶出速率慢的问题,达到增加其药理作用的效果,一方面,纳米乳因有良好的亲水亲油性,具有良好的渗透性,可以透过皮肤角质层到达目标区域,同时,通过成分配比筛选得到的纳米乳可以有效增加活性成分溶解度;另一方面,因其具有良好的促进毛发生长的作用,通过滋养毛囊可以达到抑制脱发的目的,并且通过配方的优化,使得纳米乳的制备过程简单,对仪器设备和反应条件要求低,且环境友好。采用伪三元相图法,以纳米乳区域面积为指标考察油相、表面活性剂与助表面活性剂的选择,对纳米乳进行处方筛选。选择中等脂肪链长度的甘油三酯类油相,其具有良好的流动性、溶解性能及乳化性能,可以增加体系的稳定性;选择毒性与刺激性都较小的非离子型表面活性剂与具有增溶作用的短链醇助表面活性剂联合使用,更好的降低体系界面张力促进纳米乳的形成。
因此,本发明结合中医药理论和纳米乳制剂技术,在基于中医药理论对于治疗脱发的传统中药经典组方深入分析的基础上,利用黄芩苷抗氧化、抗炎、抗病毒、促进毛发生长,苦参碱清热解毒、抗炎以及人参提取物(ginseng extract)补气益血,可以促进头部血液循环,使气血能够滋养发根,促进毛发生长等功能,研制了一款可以包载三种植物天然活性成分的纳米乳(BGM NE),纳米乳包载既解决一些活性成分溶解度低的问题,同时纳米乳的皮肤渗透性可以促进皮肤和毛囊的吸收,更好的发挥防脱发作用。
有益效果:与现有技术相比,本发明具有如下显著优点:(1)本发明的含有天然活性成分的水包油型纳米乳配方合理,安全性高,对肌肤无刺激,具有良好载药性和包封效果,载药率最高可达3.76±0.21%;(2)本发明含有天然活性成分的水包油型纳米乳的制备方法,采用环境友好的材料,且在常温常压环境下即可制得,工艺简单,生产成本低。
附图说明
图1为实施例1制备所得纳米乳的显微镜检测;
图2为实施例2制备所得纳米乳的显微镜检测;
图3为实施例3制备所得纳米乳的显微镜检测;
图4为长期稳定性试验的纳米乳粒径、pH和纳米乳中黄芩苷和苦参碱含量的变化情况;
图5为阳性对照物VC和纳米乳的DPPH自由基清除率;
图6为纳米乳的DPPH自由基清除率;
图7为阳性对照物VC的O2 -·清除率;
图8为纳米乳的O2 -·清除率;
图9为观察所得小鼠的毛发生长变化;
图10为测量的小鼠毛发生长长度情况;
图11为测量的小鼠毛发重量情况;
图12为小鼠皮肤的HE染色切片图;
图13为测量的小鼠皮肤温度变化情况。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例中人参皂苷、黄芩苷、苦参碱购自西安瑞林生物科技有限公司。
实施例1
一种含有天然活性成分的水包油型纳米乳,成分及含量如下:
表面活性剂6%,肉豆蔻酸异丙酯38%,黄芩苷1%,人参皂苷1%,苦参碱1%,泛醇0.1%,赖氨酸0.1%,苏氨酸0.1%,锌盐0.1%,防腐剂0.1%,余量为去离子水;
表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂由聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚复配而成,聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚的质量比为1:1,Km=1:1。
纳米乳的制备方法包括如下步骤:
(1)分别称取0.6mg的聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚,混合加热至35℃,加入1.2mg的无水乙醇,搅拌溶解均匀,放置室温,作为表面活性剂;
(2)按配方比例,将0.4mg黄芩苷加入15.2mg肉豆蔻酸异丙酯中,搅拌均匀,加入到表面活性剂搅拌均匀后,放至室温,作为混合物A;
(3)称取0.4mg的人参皂苷和苦参碱,0.04mg的泛醇、苏氨酸和赖氨酸加入到蒸馏水中,作为混合物B;
(4)将混合物B加入混合物A中,并加入0.04mg的苯氧乙醇并持续搅拌,直至得到透明或半透明状显蓝色乳光的纳米乳。
实施例2
一种含有天然活性成分的水包油型纳米乳,成分及含量如下:
表面活性剂8%,肉豆蔻酸异丙酯24%,黄芩苷1%,人参皂苷1%,苦参碱1%,泛醇0.1%,赖氨酸0.1%,苏氨酸0.1%,锌盐0.1%,防腐剂0.1%,余量为去离子水;
表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂由聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚复配而成,聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚的质量比为1:1,Km=2:1。
纳米乳的制备方法包括如下步骤:
(1)分别称取0.4mg的聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚,混合加热至37℃,加入0.4mg的无水乙醇,搅拌溶解均匀,放置室温,作为作为表面活性剂;
(2)按配方比例,将0.15mg黄芩苷加入3.6mg肉豆蔻酸异丙酯中,搅拌均匀,加入到表面活性剂搅拌均匀后,放至室温,作为混合物A;
(3)称0.15mg的人参皂苷和苦参碱,0.015mg的泛醇、苏氨酸和赖氨酸加入到蒸馏水中,作为混合物B;
(4)将混合物B加入混合物A中,并加入0.015mg的苯氧乙醇并持续搅拌,直至得到透明或半透明状显蓝色乳光的纳米乳。
实施例3
一种含有天然活性成分的水包油型纳米乳,成分及含量如下:
表面活性剂12%,肉豆蔻酸异丙酯18%,黄芩苷1%,人参皂苷1%,苦参碱1%,泛醇0.1%,赖氨酸0.1%,苏氨酸0.1%,锌盐0.1%,防腐剂0.1%,余量为去离子水;
表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂由聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚复配而成,聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚的质量比为1:1,Km=3:1。
纳米乳的制备方法包括如下步骤:
(1)分别称取0.6mg的聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚,混合加热至30~37℃,加入0.4mg的无水乙醇,搅拌溶解均匀,放置室温,作为表面活性剂;
(2)按配方比例,将0.13mg黄芩苷加入2.34mg肉豆蔻酸异丙酯中,搅拌均匀,加入到表面活性剂搅拌均匀后,放至室温,作为混合物A;
(3)称取0.13mg的人参皂苷和苦参碱,0.013mg的泛醇、苏氨酸和赖氨酸加入到蒸馏水中,作为混合物B;
(4)将混合物B加入混合物A中,并加入0.013mg的苯氧乙醇并持续搅拌,直至得到透明或半透明状显蓝色乳光的纳米乳。
对比例1
调整主表面活性剂与助表面活性剂的质量比制得纳米乳,成分及含量如下:
表面活性剂4%,肉豆蔻酸异丙酯24%,黄芩苷1%,人参皂苷1%,苦参碱1%,泛醇0.1%,赖氨酸0.1%,苏氨酸0.1%,锌盐0.1%,防腐剂0.1%,余量为去离子水;
表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂由聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚复配而成,聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚的质量比为1:1,Km=1:2。
纳米乳的制备方法包括如下步骤:
(1)分别称取0.2mg的聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚,混合加热至30~37℃,加入0.8mg的无水乙醇,搅拌溶解均匀,放置室温,作为表面活性剂;
(2)按配方比例,将0.3mg黄芩苷加入7.2mg肉豆蔻酸异丙酯中,搅拌均匀,加入到表面活性剂搅拌均匀后,放至室温,作为混合物A;
(3)称取处方量的0.3mg的人参皂苷和苦参碱,0.03mg的泛醇、苏氨酸和赖氨酸加入到蒸馏水中,作为混合物B;
(4)将混合物B加入混合物A中,并加入0.03mg的苯氧乙醇并持续搅拌,直至得到透明或半透明状显蓝色乳光的纳米乳。
对比例2
调整主表面活性剂与助表面活性剂的质量比制得纳米乳,成分及含量如下:
表面活性剂16%,肉豆蔻酸异丙酯24%,黄芩苷1%,人参皂苷1%,苦参碱1%,泛醇0.1%,赖氨酸0.1%,苏氨酸0.1%,锌盐0.1%,防腐剂0.1%,余量为去离子水;
表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂由聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚复配而成,聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚的质量比为1:1,Km=4:1。
纳米乳的制备方法包括如下步骤:
(1)分别称取0.8mg聚氧乙烯氢化蓖麻油40与蓖麻油聚氧乙烯醚,混合加热至30~37℃,加入0.4mg的无水乙醇,搅拌溶解均匀,放置室温,作为表面活性剂;
(2)按配方比例,将0.125mg黄芩苷加入3mg肉豆蔻酸异丙酯中,搅拌均匀,加入到表面活性剂搅拌均匀后,放至室温,作为混合物A;
(3)称取0.125mg的人参皂苷和苦参碱,0.0125mg的泛醇、苏氨酸和赖氨酸加入到蒸馏水中,作为混合物B;
(4)将混合物B加入混合物A中,并加入0.0125mg的苯氧乙醇并持续搅拌,直至得到透明或半透明状蓝色乳光的纳米乳。
针对本发明水包油型纳米乳进行试验:
电镜观察纳米乳形貌:取实施例1~3制得的水包油型纳米乳,采用透射电子显微镜,对纳米乳进行形态学评价。过0.22μm的微孔滤膜,取10μL涂于硅片表面,室温静置干燥后,用原子力显微镜进行检测。
结果如图1~3所示,纳米乳呈现类圆形结构。
检测实施例1~3和对比例1~2的粒径:
分别取实施例1~3和对比例1~2制得的纳米乳,加入一定量的蒸馏水稀释,混合均匀。在室温下,使用DLS粒径仪测定纳米乳的粒径、Zeta电位和多分散度(PDI)。
测试结果:结果如表1所示,纳米乳粒径在Km=1:1时最小,PDI也最小。表明Km的适当比例对于纳米乳液的粒径及分散度有一定影响,这可能是由于添加适当比例的助表面活性剂导致油相在表面活性剂单体的疏水区域中的更大渗透,从而进一步降低界面张力,导致界面的流动性增加,从而增加系统的熵,所形成的纳米乳尺寸更均一。
表1、纳米乳粒径和相关参数测定结果
检测实施例1~3和对比例1~2制得材料的包封率:
黄芩苷的含量可以使用紫外分光光度计在280nm波长下测得,用95乙醇配置不同浓度的纳米乳溶液,得到波长在280nm处的黄芩苷浓度与吸光度值之间的比例关系,绘制标准曲线,回归方程:y=0.3524x+0.4213(x为纳米乳的浓度,y为纳米乳溶液在280nm处的吸光度值,回归系数0.9996)。取实施例1~3和对比例1~2制得的纳米乳,取适量蒸馏水进行稀释至定量,充分混合后过0.45μm微孔滤膜,加入适量甲醇破乳,超声5min,进行吸光度测定。
计算公式:包封率=纳米乳中包裹的药物量/药物加入量×100%。
测试结果:结果如表2所示,实施例1~3的纳米乳的平均包封率为85.55%,表明纳米乳能够较好的包封黄芩苷,包封条件比较合适。而相较于实施例,对比例的纳米乳的平均包封率偏低,为70.00%,表明Km过大或过小会影响纳米乳的乳化过程,使其不易在油水界面形成一个较稳定的界面膜来促进纳米乳的形成。
表2、包封率测试结果
检测实施例1~3和对比例1~2制得材料的载药量:
将实施例1~3制得的纳米乳及对比例1~2制得的纳米乳,取适量蒸馏水进行稀释至定量,充分混合后过0.45μm微孔滤膜,加入适量甲醇破乳,超声5min,后用紫外分光光度计在280nm波长下进行吸光度测定。
计算公式:载药量=纳米乳中包裹的药物质量/纳米乳的重量×100%
测试结果:结果如表3所示,实施例的纳米乳的平均载药量为3.59%,对比例的平均载药量为2.85%。对比例的载药量均低于实施例的纳米乳载药量,由此可见,Km的量过大或过小都会影响油水界面的成膜乳化过程,降低纳米乳的稳定性或使其不易成型。
表3、载药量测试结果
从以上试验结果中得到,实施例1~3在形态、粒径、包封率和载药量上都有较好的结果,其中实施例1的粒径及包封都最好,最终选择实施例1为最佳配比。
检测实施例1~3和对比例1~2制得材料的稳定性:
分别进行了短期和长期稳定性试验。精密量取2mL的纳米乳密封于锥形瓶中,一份离心(10000r/min,10min)后,通过观察纳米乳是否出现相分离、浑浊和沉淀等外观变化来考察其稳定性。一份常温条件下(25℃)放置,分别于1、15、30天取出样品,观察纳米乳的外观性状,测定纳米乳的粒径、pH值,对纳米乳中黄芩苷和苦参碱含量进行测定来考察其稳定性。
测试结果:纳米乳在10000r·min-1离心10分钟后没有显示任何相分离、混浊和沉淀。同时,室温下1、15、30天,外观、粒径、pH、和含量均无显着差异,结果如图4所示,证明纳米乳具有良好的稳定性。
DPPH自由基清除实验:
实验方法:称取适量DPPH试剂,用无水乙醇溶解于15mL容量瓶中,定容,配制成浓度为0.115mmol/L DPPH溶液,备用。配置不同浓度的纳米乳溶液或稀释浓度的纳米乳溶液2.0mL.VC溶液配制,配制成浓度为0.001、0.005、0.01、0.02、0.05μg/mL的溶液,备用。各组分别加入之前配制的DPPH溶液2.0mL,涡旋混匀后室温下放置30min,于波长517nm处测定紫外吸光度。同样的条件下,以纯化水代替纳米乳样品作为空白对照,VC与配制的DPPH溶液反应测得结果作为阳性对照组。每组测定分别进行三次,取平均值。DPPH自由基清除率计算公式如下:
DPPH自由基清除率(%)=(1-(A1-A0)/A2)×100%
上式中A1为2.0mL纳米乳溶液与2.0mLDPPH溶液的吸光度,A0为2.0mL纳米乳溶液与2.0mL无水乙醇的吸光度,A2为2.0mL纯化水与2.0mLDPPH溶液的吸光度。
实验结果:阳性对照物VC和纳米乳的DPPH自由基清除率分别如图5和图6所示:VC在一定浓度范围内,其清除DPPH自由基的能力随着浓度增加而提升,最终在0.08μg/mL左右达到峰值,DPPH清除率为97.95%;纳米乳同样表现出良好的DPPH自由基清除能力,在一定浓度范围内,清除能力随浓度增加而提升至97.12%。
超氧阴离子自由基清除率实验:
实验方法:采用邻苯三酚氧化法,分别配制50mmol/L(pH=8.2)的Tris-HCI缓冲液、0.1mol/L的HCl溶液、25.0mmol/L的邻苯三酚溶液,放置待用。然后取5mL的试剂Tris-HCI缓冲液于试管中,在25℃水浴20min,接着加入1.0mL的样品溶液、1.0mL 25.0mmol/L的邻苯三酚溶液,混匀,25℃水浴5min后,此时再加入1.0mLHCl溶液使溶液呈酸性,在325nm下测定吸光值A。同样条件下,用蒸馏水代替样品溶液测定吸光值A0。VC反应测得结果作为阳性对照组。每组测定分别进行三次,取平均值。O2 -·的清除能力以清除率R表示。计算公式如下:
实验结果:阳性对照物VC和纳米乳的O2 -·清除率分别如图7和图8所示:VC在一定浓度范围内,其清除O2 -·的能力随着浓度增加而提升,最终在0.21μg/mL左右达到峰值,O2 -·清除率为97.87%;纳米乳表现出相对良好的O2 -·清除能力,与相同浓度下的VC对比,稍微有一定的差距,在一定浓度范围内,清除能力随浓度增加而提升至79.48%。
皮肤刺激性测试:
测试方法:试验前24h,将实验动物背部脊柱两侧毛剪掉,不可损伤表皮,去毛范围左、右各约3cm×3cm。取受试物约0.5mL(g)直接涂布于兔子背部皮肤左侧皮肤上,然后用二层纱布(2.5cm×2.5cm)和一层玻璃纸或类似物覆盖,再用无刺激性胶布和绷带加以固定,另一侧皮肤作为对照。采用封闭试验,敷用时间为4h。试验结束后用温水或无刺激性溶剂清除残留受试物。于清除受试物后的第1、24、48和72h观察涂抹部位的皮肤反应,按《化妆品卫生规范》(2007)皮肤刺激性试验中的方法进行评分和刺激强度分级(积分值小于0.5为无刺激性),计算每天每只动物的平均积分,判定皮肤刺激强度,出现皮肤刺激强度为中刺激性及以上的化妆品均判定为不合格产品。动物积分平均值按下式计算:
动物积分平均值=∑红斑和水肿积分/受试动物数
测试结果:涂抹纳米乳的每天每只动物积分值为0.24,小于0.5,表明纳米乳对皮肤无刺激性。
促毛发生长作用测试:
测试方法:取6~8周龄,18~20g,雌性C57BL/6J小鼠24只,背部用电推剪剪短毛发,在剪发区域均匀涂抹适量8%硫化钠溶液去除残余毛发,2~3min后,用温水洗净并擦干,涂抹面积约3cm×2cm。以小鼠背部皮肤光滑,无破损、无残毛为净。之后用75%乙醇对脱毛区域及周围部分消毒。选择皮肤粉红,毛发属于休止期的小鼠,建立小鼠脱发模型。脱毛次日,将小鼠随机分为4组,每组6只,分别为A组模型组,造模后不给药,给予生理盐水;B组游离药物组;C组阳性对照组(5%米诺地尔酊);D组纳米乳组。在小鼠背部脱毛区各组分别进行给药涂抹,每日1次,用量为0.3mL,覆盖脱毛区域,连续涂抹18d,观察小鼠毛发变化。
(1)小鼠毛发生长状况的观察
小鼠脱毛造模成功后,按照2分组进行给药,每天观察小鼠皮肤颜色,记录小鼠皮肤颜色变化时间和状况,并进行拍照记录。
(2)小鼠毛发生长长度的测量
观察到小鼠背部脱毛部位再次生长出毛发开始,每隔1天测量一次各组小鼠的毛发长度,一直持续至试验结束。在各组小鼠背部脱毛部位按照五点法,用镊子取背部十撮毛(每点取两撮,每个部位拔毛5~10根),用游标卡尺测量毛长,以毛发两端的最远距离记为毛发长度,得到各组小鼠毛长均值。
(3)小鼠毛发重量的测量
在试验结束时将各组小鼠处死,用电推剪尽量紧贴小鼠皮肤剪去毛发,收集各组小鼠毛发,电子分析天平称量记录,用自封袋标号保存,用十万分之一电子天平称量计算出各组小鼠毛发重量均值。
(4)小鼠皮肤HE染色考察
在脱毛后第18天颈椎脱臼法处死各组小鼠,用剪刀在C57BL/6小鼠脱毛部位剪短小鼠重新生长出的毛发,后续可再用电推剪尽量紧贴小鼠皮肤剪去毛发,在此过程中注意不要损伤小鼠皮肤。在小鼠尾部剪开,沿着脱毛区域剪下相应小鼠皮肤,将小鼠皮肤平整的贴在滤纸上,沿着脊椎平行取材,然后将皮肤修剪为1cm宽的长条状,4%多聚甲醛室温固定24h,取出固定的皮肤70%乙醇冲去多聚甲醛,梯度酒精脱水,石蜡包埋,切片,切片厚度为5μm,石蜡切片用苏木素染色10min,超纯水润洗洗去苏木素与浮色,之后伊红溶液再染色10min,梯度乙醇脱水及二甲苯透明,中性树脂对切片封固。利用正置显微镜对小鼠皮肤H&E的染色情况进行观察。每个样本随机选取3个视野进行测定和计数。实验结果以平均值±标准差表示,利用统计学软件进行统计分析,采用单因素方差分析,P<0.05则认为有统计学差异。
(5)小鼠皮肤温度变化的考察
在实验过程中,采用红外热成像仪分别测量各组小鼠在给药前后不同时间点(0min,15min,30min,60min,120min)时的皮肤温度,对比分析,考察纳米乳对于小鼠皮肤温度的影响,实验过程在恒温恒湿的动物房中进行。
测试结果:
(1)小鼠毛发生长状况的观察
结果如图9所示,观察到各组小鼠脱毛后,背部皮肤呈现粉红色,皮肤表面光滑平整,此时毛囊处于休止期;一周后小鼠皮肤颜色由粉色转变成灰色,在12d时逐渐变成灰黑色,游离组、阳性对照组和试验组毛发出现明显生长,在第15d时各组小鼠毛发均生长出毛发,模型组和游离组小鼠部分区域毛发生长缓慢,阳性对照组和试验组小鼠毛发生长更加均匀,毛发密度更佳。在第18d,模型组和游离组有较小部分区域毛发生长缓慢,阳性对照组和试验组小鼠脱毛区域毛发已经完全覆盖。
(2)小鼠毛发生长长度的测量
结果如图10所示,分别在第9、12、15和18天测量小鼠毛发长度,游离组、阳性对照组、试验组与模型组相比均有显著性差异(P<0.05),表明均有促毛发生长的作用。随着时间持续,阳性对照组与试验组的小鼠毛发生长速度相较于模型组和游离组有着明显提高,并且试验组相比游离组小鼠毛发长度明显更长(P<0.01),表明天然活性成分通过纳米乳包裹后促进皮肤吸收,发挥更好的促毛发生长作用。5%米诺地尔与试验组生长速度比较接近,但在相同时期毛发长度均低于试验组。游离成分组相比较模型组在不同时期毛发长度更长(P<0.05),但相较于试验组小鼠毛发长度偏短(P<0.01),表明天然活性成分通过纳米乳包裹后促进皮肤和毛囊吸收,发挥出更好的促毛发生长作用。
(3)小鼠毛发重量的测量
结果如图11所示,游离组与模型组相比有明显显著性差异(P<0.01),阳性对照组、试验组与模型组相比均有显著性差异(P<0.001)表明均有促毛发生长的作用。
(4)小鼠皮肤HE染色考察
对各组小鼠的皮肤HE染色切片镜下观察,结果如图12所示,并进行毛囊数量统计结果见表4。模型组小鼠皮肤中毛囊分布稀疏,毛囊之间间距较大,未见毛囊密集分布区域,游离组对比与模型组,毛囊数量明显增加(P<0.05),毛囊分布更均匀,阳性对照组和试验组小鼠皮肤内毛囊分布更加密集,可以观察到大量的毛囊,毛囊数量明显增加(P<0.05)毛囊间间距减小,表明阳性对照组和试验组可以促进小鼠毛发生长。各组小鼠的毛囊数量统计分析,各组间差异有统计学意义(F=17.9263,P<0.0001)。游离组、阳性对照组和试验组与模型组相比,毛囊数量在经过给药后都有提升,其中阳性对照组和试验组效果更佳。
表4、小鼠毛囊数量统计分析
(5)小鼠皮肤温度变化的考察
在实验过程中,采用红外热成像仪分别测量各组小鼠在给药前后不同时间点(0min,30min,60min,120min)时的皮肤温度变化如图13所示,结果显示在30min时,游离组和阳性对照组小鼠温度变化无显著性差异,纳米乳皮肤温度变化有显著性差异(P<0.05);在60min时,游离组和纳米乳组小鼠温度变化有显著性差异的影响,表明纳米乳可以扩张皮下毛细血管,提高皮肤温度,改善微循环,天然活性成分直接使用时,皮肤吸收较差在30min时温度变化无显著性差异,需要较长时间,所以在60min时温度变化有显著性差异(P<0.05)而纳米乳包载后促进皮肤吸收,在30min时温度变化有显著性差异(P<0.05)。在120min时,各组别均无显著性差异。
Claims (10)
1.一种含有天然活性成分的水包油型纳米乳,其特征在于,所述纳米乳包括如下重量百分含量的组分:表面活性剂2%~30%,油相5%~45%,黄芩苷1~2%,人参皂苷1~2%,苦参碱1~2%,泛醇0.01%~1%,赖氨酸0.05%~2%,苏氨酸0.1%~0.3%,锌盐0.05%~0.3%,防腐剂0.1%~1%,余量为水。
2.根据权利要求1所述的水包油型纳米乳,其特征在于,所述纳米乳的乳滴粒径为1~200nm。
3.根据权利要求1所述的水包油型纳米乳,其特征在于:所述油相为肉豆蔻酸异丙酯、肉豆蔻酸肉豆蔻醇酯、棕榈酸异丙酯、亚油酸异丙酯、苯甲酸十二醇酯、异硬脂醇异硬酯酸酯、脂肪酸乳酸酯、油酸癸酯或棕榈酸辛酯。
4.根据权利要求1所述的水包油型纳米乳,其特征在于,所述防腐剂为苯氧乙醇或尼泊金甲酯。
5.根据权利要求1所述的水包油型纳米乳,其特征在于:所述表面活性剂由主表面活性剂和助表面活性剂组成,其中主表面活性剂与助表面活性剂的质量比Km为1~3:1。
6.根据权利要求5所述的水包油型纳米乳,其特征在于:所述主表面活性剂由质量比为1~3:1的聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚组成,所述主表面活性剂的亲水亲油平衡值为11.8~13.4。
7.根据权利要求5所述的水包油型纳米乳,其特征在于:所述助表面活性剂为无水乙醇或聚乙二醇400。
8.根据权利要求1所述的一种水包油型纳米乳的制法,其特征在于,包括以下步骤:
(1)取聚氧乙烯氢化蓖麻油40和蓖麻油聚氧乙烯醚,混合均匀得到主表面活性剂,主表面活性剂和助表面活性剂按照质量比1~3:1混合得到表面活性剂;
(2)将黄芩苷溶解于油相中,加入表面活性剂混合均匀得到混合物A;
(3)将人参皂苷、苦参碱、泛醇、苏氨酸和赖氨酸溶于水中,得到混合物B;
(4)将混合物B加入混合物A中,持续搅拌均匀,得到含有天然活性成分的水包油型纳米乳。
9.根据权利要求8所述的制法,其特征在于,步骤(1)中的搅拌混合温度为30~37℃。
10.一种权利要求1所述的水包油型纳米乳在制备治疗脱发、促进毛发生长的药物或洗护用品中的应用。
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