CN113101374B - 一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体及其制备方法 - Google Patents
一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体及其制备方法 Download PDFInfo
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- CN113101374B CN113101374B CN202110379790.9A CN202110379790A CN113101374B CN 113101374 B CN113101374 B CN 113101374B CN 202110379790 A CN202110379790 A CN 202110379790A CN 113101374 B CN113101374 B CN 113101374B
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- seed oil
- peony seed
- gelatin
- liposome
- acacia
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Abstract
本发明属于纳米脂质体技术领域,特别涉及一种阿拉伯胶‑明胶修饰牡丹籽油纳米脂质体的制备方法,首次采用明胶和阿拉伯胶以逐层静电沉积技术对纳米脂质体进行修饰,并对制备工艺进行优化,将溶液的pH始终控制在4.5~5,明胶和阿拉伯胶在最终混合体系中质量浓度控制在0.2%,使得制备得到的阿拉伯胶‑明胶修饰的脂质体具有较未修饰脂质体更高的包封率、更强的抗氧化性、更好的贮存稳定性、更好的消化稳定性和更好的热稳定性。同时与现有技术阿拉伯胶‑壳聚糖修饰脂质体对比发现,阿拉伯胶‑明胶修饰得到的脂质体粒径更小、抗氧化更好、热稳定性更好。
Description
技术领域
本发明属于纳米脂质体技术领域,特别涉及一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体及其制备方法。
背景技术
纳米脂质体是应用最广泛的封装和药物递送系统之一。它们是脂质体的纳米级版本,类似于球形囊泡,由围绕水核的一个或多个磷脂双层膜组成,其中磷脂分子由胆碱,磷酸酯和甘油组成的极性头基(亲水性),具有亲水性,以及由疏水性的长烃链组成的非极性尾基(疏水性)。由于纳米脂质体囊泡既具有水相又具有脂质相,因此它们可以轻松地包裹疏水,亲水或两亲性分子。亲水性和亲脂性分子可以分别包封在内部水相和脂质体载体的脂质双层之间的区域中。同时,两亲分子可以被困在水/脂质相中。该递送系统的主要障碍是稳定性较低和封装药物的泄漏。这是主要是由于低pH和酶促条件引起的磷脂水解和储存过程中的氧化反应导致磷脂双层损伤。迄今为止,通过生物聚合物的缀合修饰脂质体表面可以减少磷脂的氧化损伤和水解降解。生物聚合物的共轭还可以使磷脂和生物聚合物分子之间形成静电桥,这可以使磷脂双层的渗透性最小化,从而增强载药脂质体系统的稳定性。目前已有使用壳聚糖和阿拉伯胶对脂质体进行单层或多层修饰的技术,但修饰后的脂质体粒径过大,并且耐高温性和氧化稳定性较差,所以需要寻求一种新的材料对脂质体进行修饰。
发明内容
本发明的目的在于提供一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体及其制备方法,解决了现有修饰后的脂质体粒径过大,并且耐高温性和氧化稳定性较差的问题。
本发明是通过以下技术方案来实现:
一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,包括以下步骤:
S1、制备牡丹籽油纳米脂质体分散液,并冷藏保存;
S2、将pH值为4.5~5明胶溶液加热,将牡丹籽油纳米脂质体分散液滴入明胶溶液中,搅拌后,带正电的明胶沉积在带负电的牡丹籽油纳米脂质体表面,得到带正电的明胶-牡丹籽油纳米脂质体溶液,然后调节pH至4.5~5;
S3、将pH值为4.5~5的带负电的阿拉伯胶溶液加入明胶-牡丹籽油纳米脂质体溶液中,搅拌均匀,得到混合体系,将混合体系的pH值调至4.5~5,得到阿拉伯胶-明胶修饰牡丹籽油纳米脂质体;
其中,混合体系中,明胶和阿拉伯胶的质量浓度均为0.2%。
进一步,明胶溶液的制备过程为:将明胶溶于蒸馏水中,溶解后,调节pH至4.5~5。
进一步,阿拉伯胶溶液的制备过程为:将阿拉伯胶溶于蒸馏水中,溶解后,调节pH至4.5~5。
进一步,阿拉伯胶溶液、明胶溶液、牡丹籽油脂质体溶液的体积比为1:1:1。
进一步,S2中,加热具体为:水浴加热至45℃。
进一步,搅拌时间为20min。
进一步,牡丹籽油纳米脂质体分散液的制备过程为:
S1.1、称取蛋黄卵磷脂、胆固醇及牡丹籽油,蛋黄卵磷脂、胆固醇及牡丹籽油的质量比为6:1:1;
S1.2、将蛋黄卵磷脂和胆固醇混合搅拌,得到油相I,降温至室温;将牡丹籽油加入油相I中,混合得到油相Ⅱ,升温后搅拌,然后降温至室温;
取甘油加入蒸馏水中,预热后得到水相;
S1.3、将水相加入油相Ⅱ,混合搅拌,然后孵育,得到混合液;之后将混合液在高压条件下均化后得到初乳,降温至0~4℃;
S1.4、将初乳进行超声分散,得到牡丹籽油纳米脂质体分散液。
进一步,S1.4中,以10000r/min的转速均化。
进一步,S1.4中,超声分散的参数具体为:以233W的功率、1s/2s模式超声6min。
本发明还公开了所述的制备方法制备得到的阿拉伯胶-明胶修饰牡丹籽油纳米脂质体。
与现有技术相比,本发明具有以下有益的技术效果:
本发明公开了一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,首次采用明胶和阿拉伯胶以逐层静电沉积技术对纳米脂质体进行修饰,并对制备工艺进行优化,将溶液的pH始终控制在4.5~5,明胶和阿拉伯胶在最终混合体系中质量浓度控制在0.2%,使得制备得到的阿拉伯胶-明胶修饰的脂质体具有较未修饰脂质体更高的包封率、更强的抗氧化性、更好的贮存稳定性、更好的消化稳定性和更好的热稳定性。同时与现有技术阿拉伯胶-壳聚糖修饰脂质体对比发现,阿拉伯胶-明胶修饰得到的脂质体粒径更小、抗氧化更好、热稳定性更好。
进一步,本发明采用热熔法制备牡丹籽油纳米脂质体,不使用有机溶剂,避免有机溶剂的残留对细胞产生毒性;本发明采用多次升温-降温的方法制备脂质体,可以避免油脂由于持续加热导致氧化,并且得到的脂质体包封率高于未改良的、持续加热的热熔法制备得到的脂质体;本发明在热熔法基础上采用均化和超声技术联合使用的方法制备纳米脂质体,可以起到充分分散脂质体的效果,有效减小脂质体的粒径。热熔法是一种不需要使用有机溶剂的制备脂质体的方法,其原理是通过加热使温度达到表面活性剂相变温度以上,从而使蛋黄卵磷脂等的双层膜通透性和流动性升高,从而使被包封物质可以掺入至脂双层中,之后水相的加入使得蛋黄卵磷脂在水相中自组装形成球状脂质体囊泡,之后通过磁力搅拌的方式使脂质体稳定,并起到分散脂质体减小粒径的目的。
附图说明
图1为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体在不同放大倍数下的透射电子显微照片;
图2为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体在60℃下储存15天的氢过氧化物浓度的变化曲线;
图3为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体在40℃下储存15天的氢过氧化物浓度的变化曲线;
图4为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体在60℃下储存15天的TBARS浓度的变化曲线;
图5为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体在40℃下储存15天的TBARS浓度的变化曲线;
图6为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体在60℃下储存15天的包封率的变化曲线;
图7为牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体在40℃下储存15天的包封率的变化曲线。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
阿拉伯胶是一种阴离子多糖,具有良好的水溶性和乳化性。阿拉伯胶由70%的多糖和少量的蛋白组成。其中多糖以共价键与蛋白肽链中的羟脯氨酸、丝氨酸结合,与蛋白质相连接的多糖分子是高度分支的酸性多糖。它由D-半乳糖、L-阿拉伯糖、D-葡萄糖醛酸、L-鼠李糖、4-O-甲基-D-葡萄糖醛酸几部分组成。因为阿拉伯胶结构上带有酸性基团,溶液的自然pH值也呈弱酸性,具有酸环境较稳定的特性。与使用单一生物聚合物相比,生物聚合物的络合(例如通过化学反应使壳聚糖和阿拉伯胶结合)可以改善胃肠道中的药物递送和控释特性。
蛋白质和多糖也可以产生生物聚合物,明胶具有生物相容性,可生物降解,可食用并且在人体温度下可溶,因此是食品和制药应用的理想材料。明胶在其等电点以下带正电,可以与阿拉伯胶在其等电点以下的pH值范围内可形成聚电解质复合物。在蛋白质和多糖复合物形成的过程中具有若干关键pH转折点。它们分别对应于不同反应程度的复合物,包括可溶性复合物、不可溶性复合物及凝聚物。经过研究得到明胶-阿拉伯胶体系存在可溶性复合物阶段,在该阶段产生的聚合物具有纳米级尺寸,而不可溶性复合物和凝聚物阶段产生的聚合物尺寸均达到微米级及以上,所以为了使明胶-阿拉伯胶-纳米脂质体粒径在纳米级范围内,需要在明胶-阿拉伯胶体系在可溶性复合物阶段的pH值范围内进行纳米脂质体的修饰。
且明胶在蛋白质中具有较好的耐高温性,所以本发明采用明胶-阿拉伯胶双层修饰脂质体,并与壳聚糖-阿拉伯胶双层修饰脂质体进行理化性质对照。
基于单因素实验确定最佳修饰工艺后,确定阿拉伯胶与壳聚糖最佳修饰浓度为0.2%(w/w),最佳修饰质量比为CS:GA=1:1,最佳修饰pH为4,阿拉伯胶与明胶最佳修饰浓度为0.2%(w/w),最佳修饰质量比为GE:GA=1:1,最佳修饰pH为4.5-5。
实施例1
本发明公开了一种抗氧化纳米牡丹籽油纳米脂质体的制备方法,包括以下步骤:
1、制备牡丹籽油纳米脂质体分散液,制备过程为:
取蛋黄卵磷脂0.3g,胆固醇0.05g,置于恒温磁力搅拌器上在50℃下以600r/min转速混合20min搅拌均匀,得到油相I,降温至室温。
取牡丹籽油0.06g,加入油相I,升温至50℃,混合20min,得到油相Ⅱ,降温至室温。取甘油3ml加入150ml蒸馏水中,预热至50℃,得到水相。
将水相加入油相Ⅱ,在磁力搅拌器上以50℃、600r/min转速混合10min,孵育30min。之后将混合液以10000r/min的转速均化2min,得到初乳。
将初乳通过探头超声以233W的功率、1s/2s模式超声6min,得到牡丹籽油纳米脂质体分散液,将制备得到的牡丹籽油纳米脂质体分散液储存在4℃冰箱中备用。
2、明胶溶液、阿拉伯胶溶液制备
将0.6g明胶溶于100mL的蒸馏水中得到明胶溶液,将明胶溶液过夜搅拌使其充分溶解,将其pH调至4.5。
将0.6g的阿拉伯胶溶于100mL的蒸馏水中得到阿拉伯胶溶液,将阿拉伯胶溶液过夜搅拌使其充分溶解,将阿拉伯胶溶液pH调至4.5。
3、阿拉伯胶与明胶层层自组装修饰牡丹籽油纳米脂质体
生物聚合物在纳米脂质体表面的结合是通过静电逐层自组装方法分两步进行的,首先将明胶溶液水浴加热至45℃,将牡丹籽油纳米脂质体分散液滴入明胶溶液中,磁力搅拌20min,使带正电的明胶沉积在带负电的脂质体表面,得到明胶-牡丹籽油纳米脂质体溶液,然后调节pH至4.5。
使用注射器将带负电的阿拉伯胶溶液注入带正电的明胶-纳米脂质体溶液中,同时磁力搅拌20min,以形成均匀的阿拉伯胶-明胶-牡丹籽油纳米脂质体溶液。将所得的阿拉伯胶-明胶-牡丹籽油纳米脂质体溶液的pH值调至4.5,得到阿拉伯胶-明胶修饰牡丹籽油纳米脂质体,储存在4℃冰箱中。
实施例2
本发明公开了一种抗氧化纳米牡丹籽油纳米脂质体的制备方法,包括以下步骤:
1、制备牡丹籽油纳米脂质体分散液,制备过程同实施例1。
2、明胶溶液、阿拉伯胶溶液制备
将0.6g明胶溶于100mL的蒸馏水中得到明胶溶液,将明胶溶液过夜搅拌使其充分溶解,将其pH调至5。
将0.6g的阿拉伯胶溶于100mL的蒸馏水中得到阿拉伯胶溶液,将阿拉伯胶溶液过夜搅拌使其充分溶解,将阿拉伯胶溶液pH调至5。
3、阿拉伯胶与明胶层层自组装修饰牡丹籽油纳米脂质体
生物聚合物在纳米脂质体表面的结合是通过静电逐层自组装方法分两步进行的,首先将明胶溶液水浴加热至45℃,将牡丹籽油纳米脂质体分散液滴入明胶溶液中,磁力搅拌20min,使带正电的明胶沉积在带负电的脂质体表面,得到明胶-牡丹籽油纳米脂质体溶液,然后调节pH至5。
使用注射器将带负电的阿拉伯胶溶液注入带正电的明胶-纳米脂质体溶液中,同时磁力搅拌20min,以形成均匀的阿拉伯胶-明胶-牡丹籽油纳米脂质体溶液。将所得的阿拉伯胶-明胶-牡丹籽油纳米脂质体溶液的pH值调至5,得到阿拉伯胶-明胶修饰牡丹籽油纳米脂质体,储存在4℃冰箱中。
阿拉伯胶溶液、明胶溶液、牡丹籽油脂质体溶液的体积比控制为1:1:1,方便计算,方便将混合体系中明胶和阿拉伯胶的质量浓度控制为0.2%。
对照例1
1、制备牡丹籽油纳米脂质体分散液,同实施例1的过程。
2、壳聚糖溶液、阿拉伯胶溶液制备
将0.6g壳聚糖溶于100mL1%的乙酸中,制备得到壳聚糖溶液,将壳聚糖溶液过夜搅拌使其充分溶解,将其pH调至4。
将0.6g的阿拉伯胶溶于100mL的蒸馏水中得到阿拉伯胶溶液,将阿拉伯胶溶液过夜搅拌使其充分溶解,将阿拉伯胶溶液pH调至4。
3、阿拉伯胶与壳聚糖层层自组装修饰牡丹籽油纳米脂质体
首先将牡丹籽油纳米脂质体分散液逐滴滴入等体积的壳聚糖溶液中,使壳聚糖溶液可以沉积在带负电荷的脂质体表面,在滴加过程中采用磁力搅拌持续搅拌20min,均匀分布以形成壳聚糖-纳米脂质体,然后调节pH至4。
使用注射器将带负电的阿拉伯胶溶液注入带正电的壳聚糖-牡丹籽油纳米脂质体溶液中,同时磁力搅拌20min,以形成均匀的阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体溶液。将所得的阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体溶液的pH值调至4,储存在4℃冰箱中。
效果验证:
1.粒径、多分散系数、Zeta电位、包封率的测定
将样品用去离子水稀释10倍,防止微粒聚集影响测定结果,之后将样品装入特定样品池,采用安东帕LitesizerTM500型纳米粒度及Zeta电位分析仪测定样品的粒径分布、多分散性系数以及Zeta电位。
包封率测定方法采用有机溶剂萃取法:移取5mL牡丹籽油纳米脂质体至离心管中,加入正己烷10mL提取游离油,再将混合溶液进行离心4000rpm/min,20min。取上清液在234nm处测定吸光值A,同法萃取后测定空白脂质体的吸光值A0,计算ΔA(A-A0)。通过标曲计算游离油含量,总油含量通过初始添加油含量得到计算包封率。
EE%=(总油含量-游离油含量)/总油含量
表1列出了牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体的平均粒径,多分散指数PDI,Zeta电位,包封率。
表1未修饰和修饰的牡丹籽油纳米脂质体的平均粒径(nm),多分散指数PDI,zeta电位(mV),包封率(%)
如表1所示,牡丹籽油纳米脂质体的平均粒径为275.6±0.45nm,在将阿拉伯胶-壳聚糖修饰至牡丹籽油纳米脂质体表面后,脂质体平均粒径显著增加至587.366±32.361nm(p<0.05),在将阿拉伯胶-明胶修饰至牡丹籽油纳米脂质体表面后(pH4.5-5),脂质体平均粒径显著增至440.733±2.495-467.036±4.653nm(p<0.05),这可能归因于聚电解质之间通过静电相互作用在牡丹籽油纳米脂质体表面上形成了一层厚的层,并且带电复聚物之间桥接絮凝所致。对于修饰后的样品,修饰有阿拉伯胶-壳聚糖的牡丹籽油纳米脂质体(587.366±32.361nm)的平均粒径显著大于修饰有阿拉伯胶-明胶的牡丹籽油纳米脂质体(440.733±2.495-467.036±4.653nm)(p<0.05)。这可能是由于壳聚糖所带正电荷较多,吸附了较多的阿拉伯胶,导致形成的涂层比阿拉伯胶-明胶-牡丹籽油纳米脂质体厚,从而导致产生更大的粒径与更高的PDI。
由表1可知,经过阿拉伯胶-壳聚糖修饰后,脂质体的Zeta电位由-28.4±0.45mV转变为37.6±1.473mV,脂质体所带电荷由负电荷转变为正电荷,且Zeta电位绝对值显著增加(p<0.05)。通常,脂质体表面所带的负电荷归因于磷,而经过修饰后转变为正电荷则是由于阿拉伯胶-壳聚糖涂层成功修饰在脂质体表面。同样,经过阿拉伯胶-明胶修饰后,脂质体Zeta电位变为-11.9±0.2mV和-12.2±0.4mV,虽然由于明胶所带正电荷无法完全中和脂质体所带负电荷并将其反转为正值,但修饰后脂质体Zeta电位的绝对值显著减小(p<0.05),这说明阿拉伯胶-明胶涂层的修饰改变了脂质体表面所带电荷量。
由表1可知,经过修饰的牡丹籽油纳米脂质体的包封率提高,这是由于聚电解质在脂质体外层交联并形成三维网络,从而将部分未包封的游离油困在三维网络中,导致修饰后的脂质体包封率提高。
2.透射电子显微镜观察
采用磷钨酸负染法对牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体及阿拉伯胶-明胶-牡丹籽油纳米脂质体进行前处理,取适量样品,滴于铜网上停留4min,用滤纸吸干边缘多余的样品,1%的磷钨酸溶液染色2min,用滤纸吸干边缘多余溶液后自然晾干,用透射电镜观察脂质体的形态结构,加速电压为80kV。结果如图1所示。
如图1所示的TEM图像,从图1(a-1)和(a-2)中可看到未修饰的牡丹籽油纳米脂质体呈球形,能观察到内部具有清晰的脂质体特有指纹结构,且粒径显示约为250nm左右。由图1(b-1)和(b-2)中可看到阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体呈现球形或不规则形状,同样可以观察到内部清晰地脂质体特有指纹结构,说明经过阿拉伯胶-壳聚糖修饰后没有改变脂质体的内部结构。可以观察到纳米脂质体被白色物质(阿拉伯胶-壳聚糖涂层)围绕,粒径显示在550nm左右。
由图1(c-1)和(c-2)中可看到阿拉伯胶-明胶-牡丹籽油纳米脂质体呈现球形或不规则形态,同样可以观察到内部的指纹结构,说明经过阿拉伯胶-明胶修饰没有影响脂质体内部结构。可以观察到纳米脂质体被较浅的白色光晕(阿拉伯胶-明胶涂层)围绕,粒径显示在450nm左右。
5.加速氧化实验中氢过氧化物浓度的测定
5.1 60℃加速氧化实验中氢过氧化物浓度的测定
将新鲜制备好的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体样品置于60℃烘箱中储藏15天,测量样品的氢过氧化物浓度。取样时间为0、3、6、9、12、15d。
氢过氧化物的测定方法:用移液枪分别吸取0.3mL乳液于同样大小离心管中,分别加入1.5mL异辛烷和异丙醇的混合液(异辛烷和异丙醇混合的体积比例为3:1),充分震荡10s,重复3次,离心(1000g,2min),分别取上层有机层200μL于离心管中,分别加入2.8mL甲醇和丁醇体积比2:1的混合物,然后再分别加入15μL、3.94mol/L的硫氰酸铵,再加入15μL二价铁离子溶液(0.132mol/L氯化钡和0.144mol/L硫酸亚铁以体积比1:1的比例混合,再过0.22μm滤膜),避光反应20min后,于510nm波长下测定吸光度值,通过过氧化氢异丙苯标准曲线计算样品中氢过氧化物浓度。
将新鲜制备的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体置于60℃烘箱加速氧化,样品中氢过氧化物浓度如图2所示。由图可知,在0-6d的时间内,未修饰的牡丹籽油纳米脂质体的氢过氧化物浓度急剧增加,在6-12d内增加速率降低,在12-15d内氢过氧化物浓度下降。氢过氧化物浓度在15d内由初始的0.785±0.015mmol/L在15d时增长至8.982±0.533mmol/L,期间最高值为9.651±0.184mmol/L。经过阿拉伯胶-壳聚糖双层修饰的牡丹籽油纳米脂质体在0-15d内氢过氧化物浓度持续增长,但增加速率显著低于未修饰牡丹籽油纳米脂质体,阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体的氢过氧化物浓度由初始的0.716±0.245mmol/L增长至15d的6.99±0.315mmol/L,阿拉伯胶-明胶修饰的牡丹籽油脂质体的氢过氧化物浓度由初始的0.788±0.01mmol/L(pH4.5)和0.748±0.024mmol/L(pH5)在第15d时增长至5.379±0.034mmol/L(pH4.5)和5.479±0.465mmol/L(pH5),氢过氧化物浓度显著低于阿拉伯胶-壳聚糖修饰的牡丹籽油脂质体,说明阿拉伯胶-明胶涂层在60℃加速氧化条件下的保护效果显著好于阿拉伯胶-壳聚糖涂层。
5.2 40℃加速氧化实验中氢过氧化物浓度的测定
将新鲜制备好的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体样品置于40℃烘箱中储藏15天,测量样品的氢过氧化物浓度。取样时间为0、3、6、9、12、15d。
将新鲜制备的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体置于40℃烘箱加速氧化,样品中氢过氧化物浓度如图3所示。由图可知,与60℃加速氧化类似,在0-6d的时间内,未修饰的牡丹籽油纳米脂质体的氢过氧化物浓度急剧增加,在6-9d内增加速率降低,在9-15d内氢过氧化物浓度下降。氢过氧化物浓度在15d内由初始的0.725±0.039mmol/L在15d时增长至3.821±0.271mmol/L,期间最高值为4.298±0.305mmol/L。阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体的氢过氧化物浓度由初始的1.144±0.039mmol/L增长至15d的3.421±0.189mmol/L,阿拉伯胶-明胶修饰的牡丹籽油脂质体的氢过氧化物浓度由初始的1.229±0.024mmol/L(pH4.5)和1.125±0.112mmol/L(pH5)在第15d时增长至2.931±0.261mmol/L(pH4.5)和2.752±0.262mmol/L(pH5),氢过氧化物浓度显著低于阿拉伯胶-壳聚糖修饰的牡丹籽油脂质体,说明阿拉伯胶-明胶涂层在40℃加速氧化条件下的保护效果显著好于阿拉伯胶-壳聚糖涂层。
6.加速氧化实验中次级氧化产物—TBARS的测定
6.1 60℃加速氧化实验中次级氧化产物—TBARS的测定
将新鲜制备好的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体样品置于60℃烘箱中储藏15天,测量样品的次级氧化产物的浓度。取样时间为0、3、6、9、12、15d。
次级氧化产物浓度的测定方法:取1mL脂质体溶液加入到离心管中,再加入5mLTBA测试液(由15%的三氯乙酸和0.375%的硫代巴比妥酸溶于0.25mol/L HCl配制而成),然后将混合物在沸水(95–100℃)中加热15分钟,形成粉红色,用流动的自来水冷却,放置10min,采用1.2μm的微孔滤膜过滤,滤液在532nm下测定吸光值。次级氧化产物的浓度以1,1,3,3-四乙氧基丙烷标准曲线进行计算。
油脂次级氧化产物是由初级氧化产物进一步氧化生成醛类或酸类等小分子物质,这些物质是引起哈喇味根源,图4为在60℃下加速氧化的脂质体样品中次级氧化产物的生成情况。由图可知,与氢氧化物浓度变化情况相似,牡丹籽油纳米脂质体产生次级氧化产物的速度高于阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体。这是由于牡丹籽油纳米脂质体初级氧化产物生成速度快,积累多,所以分解成次级氧化产物的速度也快。而阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体和阿拉伯胶-明胶-牡丹籽油纳米脂质体初级氧化产物生成速度低于牡丹籽油纳米脂质体,所以次级产物累积浓度也低于牡丹籽油纳米脂质体。在15d时,牡丹籽油纳米脂质体的TBARS浓度为7.491±0.31μmol/L,阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体的TBARS浓度为5.662±0.245μmol/L,阿拉伯胶-明胶-牡丹籽油纳米脂质体的TBARS浓度为2.493±0.136μmol/L(pH4.5)和2.393±0.186μmol/L(pH5)。可知阿拉伯胶-明胶修饰后的牡丹籽油脂质体次级氧化产物产生的速度显著低于阿拉伯胶-壳聚糖修饰的脂质体,说明阿拉伯胶-明胶修饰涂层的保护效果优于阿拉伯胶-壳聚糖涂层。
6.2 40℃加速氧化实验中次级氧化产物—TBARS的测定
将新鲜制备好的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体样品置于40℃烘箱中储藏15天,测量样品的次级氧化产物的浓度。取样时间为0、3、6、9、12、15d。
图5为在40℃下加速氧化的脂质体样品中次级氧化产物的生成情况。由图可知,与60℃加速氧化样品TBARS浓度变化情况相似,40℃加速氧化下,在15d时,牡丹籽油纳米脂质体的TBARS浓度为4.579±0.124μmol/L,阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体的TBARS浓度为2.605±0.255μmol/L,阿拉伯胶-明胶-牡丹籽油纳米脂质体的TBARS浓度为2.219±0.261μmol/L(pH4.5)和2.022±0.197μmol/L(pH5)。可知阿拉伯胶-明胶修饰后的牡丹籽油脂质体次级氧化产物产生的速度显著低于阿拉伯胶-壳聚糖修饰的脂质体,说明阿拉伯胶-明胶修饰涂层的保护效果优于阿拉伯胶-壳聚糖涂层。
7.加速氧化实验中脂质体包封率的测定
7.1 60℃加速氧化实验中脂质体包封率的测定
将新鲜制备好的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体样品置于60℃烘箱中储藏15天,测量样品的包封率,按照1.的方法测量。取样时间为0、3、6、9、12、15d。
图6为样品在60℃下加速氧化时对牡丹籽油包封率的变化情况。由图可知,在0-15d的储藏期内,牡丹籽油纳米脂质体的包封率下降速度最快,由初始的95.033±1.097%降至66.967±1.115%,包封率在15d内下降了29.532%。阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体包封率下降速度小于牡丹籽油纳米脂质体,但大于阿拉伯胶-明胶-牡丹籽油纳米脂质体,从初始的96.253±1.627%降至70.531±1.986%,下降了26.723%。阿拉伯胶-明胶-牡丹籽油纳米脂质体下降速度最慢,由初始的96.1±0.802%(pH4.5)和96.247±0.522%(pH5)下降至74.367±1.06%(pH4.5)和75.571±0.231%(pH5),下降了22.615%(pH4.5)和21.482%(pH5)。说明修饰涂层的三维网络结构可以很好地保护脂质体,提高脂质体的热稳定性和氧化稳定性。阿拉伯胶-明胶涂层在热稳定性上效果优于阿拉伯胶-壳聚糖涂层,这可能是由于阿拉伯胶-明胶络合物在高温影响下解体速度慢于阿拉伯胶-壳聚糖涂层,所以在高温下对脂质体的保护效果可以持续更长时间。
7.2 40℃加速氧化实验中脂质体包封率的测定
将新鲜制备好的牡丹籽油纳米脂质体、阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体样品置于40℃烘箱中储藏15天,测量样品的包封率,按照1.的方法测量。取样时间为0、3、6、9、12、15d。
图7为样品在40℃下加速氧化时对牡丹籽油包封率的变化情况。由图可知,与60℃加速氧化结果类似,在0-15d的储藏期内,牡丹籽油脂质体的包封率由初始的93.333±0.611%降至76.411±1.405%,包封率在15d内下降了18.131%。阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体包封率从初始的95.213±1.627%降至83.733±1.155%,下降了12.057%。阿拉伯胶-明胶-牡丹籽油纳米脂质体下降速度最慢,由初始的94.102±0.803%(pH4.5)和95.247±0.522%(pH5)下降至86.5±0.889%(pH4.5)和88.202±0.231%(pH5),下降了8.078%(pH4.5)和7.397%(pH5)。阿拉伯胶-明胶涂层修饰的牡丹籽油脂质体包封率下降比率显著低于阿拉伯胶-壳聚糖涂层修饰的脂质体,说明在40℃下,阿拉伯胶-明胶涂层对脂质体的保护效果优于阿拉伯胶-壳聚糖涂层。
本发明采用逐层静电沉积(LBL)技术制备了阿拉伯胶-壳聚糖修饰的牡丹籽油脂质体和阿拉伯胶-明胶修饰的牡丹籽油脂质体,并将阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体、阿拉伯胶-明胶-牡丹籽油纳米脂质体、未修饰纳米脂质体进行对比。发现阿拉伯胶-明胶修饰的脂质体的粒径显著小于(p<0.05)阿拉伯胶-壳聚糖修饰的脂质体,并且氧化稳定性和热稳定性较阿拉伯胶-壳聚糖-牡丹籽油纳米脂质体有显著提高(p<0.05),说明阿拉伯胶-明胶涂层的保护效果优于阿拉伯胶-壳聚糖涂层。
Claims (10)
1.一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,包括以下步骤:
S1、制备牡丹籽油纳米脂质体分散液,并冷藏保存;
S2、将pH值为4.5-5明胶溶液加热,将牡丹籽油纳米脂质体分散液滴入明胶溶液中,搅拌后,带正电的明胶沉积在带负电的牡丹籽油纳米脂质体表面,得到带正电的明胶-牡丹籽油纳米脂质体溶液,然后调节pH至4.5-5;
S3、将pH值为4.5-5的带负电的阿拉伯胶溶液加入明胶-牡丹籽油纳米脂质体溶液中,搅拌均匀,得到混合体系,将混合体系的pH值调至4.5-5,得到阿拉伯胶-明胶修饰牡丹籽油纳米脂质体;
其中,混合体系中,明胶和阿拉伯胶的质量浓度均为0.2%。
2.根据权利要求1所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,明胶溶液的制备过程为:将明胶溶于蒸馏水中,溶解后,调节pH至4.5-5。
3.根据权利要求1所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,阿拉伯胶溶液的制备过程为:将阿拉伯胶溶于蒸馏水中,溶解后,调节pH至4.5-5。
4.根据权利要求1所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,阿拉伯胶溶液、明胶溶液、牡丹籽油脂质体溶液的体积比为1:1:1。
5.根据权利要求1所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,S2中,加热具体为:水浴加热至45℃。
6.根据权利要求1所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,搅拌时间为20min。
7.根据权利要求1所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,牡丹籽油纳米脂质体分散液的制备过程为:
S1.1、称取蛋黄卵磷脂、胆固醇及牡丹籽油,蛋黄卵磷脂、胆固醇及牡丹籽油的质量比为6:1:1;
S1.2、将蛋黄卵磷脂和胆固醇混合搅拌,得到油相Ⅰ,降温至室温;将牡丹籽油加入油相Ⅰ中,混合得到油相Ⅱ,升温后搅拌,然后降温至室温;
取甘油加入蒸馏水中,预热后得到水相;
S1.3、将水相加入油相Ⅱ,混合搅拌,然后孵育,得到混合液;之后将混合液在高压条件下均化后得到初乳,降温至0~4℃;
S1.4、将初乳进行超声分散,得到牡丹籽油纳米脂质体分散液。
8.根据权利要求7所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,S1.3中,以10000r/min的转速均化。
9.根据权利要求7所述的一种阿拉伯胶-明胶修饰牡丹籽油纳米脂质体的制备方法,其特征在于,S1.4中,超声分散的参数具体为:以233W的功率、1s/2s模式超声6min。
10.权利要求1-9任意一项所述的制备方法制备得到的阿拉伯胶-明胶修饰牡丹籽油纳米脂质体。
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