CN114159406A - 一种复合抗炎纳米颗粒及其制备方法与应用 - Google Patents
一种复合抗炎纳米颗粒及其制备方法与应用 Download PDFInfo
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- CN114159406A CN114159406A CN202111081920.7A CN202111081920A CN114159406A CN 114159406 A CN114159406 A CN 114159406A CN 202111081920 A CN202111081920 A CN 202111081920A CN 114159406 A CN114159406 A CN 114159406A
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- inflammatory
- acid
- burdock
- ursolic acid
- oleanolic acid
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Abstract
本发明涉及医药技术领域,具体涉及一种复合抗炎纳米颗粒及其制备方法,所纳米颗粒由熊果酸、齐墩果酸和牛蒡多糖组成,熊果酸和齐墩果酸被包覆在牛蒡多糖中;其中,熊果酸和齐墩果酸的质量比为1~3:1~3;熊果酸和齐墩果酸的质量的和与牛蒡多糖的质量比为1:10~100;本发明中发现熊果酸和齐墩果酸的抗炎活性具有协同作用,并且将牛蒡多糖ALP与熊果酸、齐墩果酸相结合,使用抗溶剂沉淀法制备了ALP‑OA/UA纳米颗粒,所获得的ALP‑OA/UA纳米粒子能有效提高OA和UA的水溶性和分散性,包封率为48.98%,负载量为0.96%;ALP‑OA/UA纳米颗粒的粒度为199.1nm,zeta电位为‑7.15mV;ALP和ALP‑OA/UA纳米颗粒可以显着抑制CuSO4引起的斑马鱼炎症反应。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种复合抗炎纳米颗粒及其制备方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
熊果酸(UA)和齐墩果酸(OA)为一对互为同分异构体的五环三萜类化合物,具有类似的分子结构和药理学活性。近年来因其具有抗炎、保肝、抗氧化以及免疫调节等多种活性被广泛报道。在所研究的这些药理活性中,抗炎活性最受关注,因为大多生物活性研究都与抗炎作用有关。而前期研究对OA和UA单独抗炎作用研究很多且主要集中于提取物和单体组分的研究,但对于二者之间存在协同作用的研究鲜见报道。并且因为UA和OA水溶性差,生物利用度低,极大的限制了其在食品及药品行业的应用。现有技术已知通过构建UA和OA的递送系统可以有效的提高其生物利用度,如:微胶囊、纳米乳液、脂质体、纳米颗粒等。纳米级形式可优化食品的理化性质,改善其营养和风味,提高活性成分的生物利用率,促进活性成分的消化吸收。纳米颗粒具有合成简便、成本较低以及生物可降解性等特点,可作为最有潜力的药物递送体系,但食品体系中的大规模表面活性剂的使用可能会对人体造成潜在损害。
发明内容
针对现有技术中存在的问题,本发明的目的是提供一种复合抗炎纳米颗粒及其制备方法与应用,本发明中研究了熊果酸和齐墩果酸的抗炎活性,发现熊果酸和齐墩果酸的抗炎活性具有协同作用,并且本发明将牛蒡多糖(ALP)与熊果酸、齐墩果酸相结合,使用抗溶剂沉淀法制备了ALP-OA/UA纳米颗粒,所获得的ALP-OA/UA纳米粒子能有效提高OA和UA的水溶性和分散性,包封率达到48.98%,负载量达到0.96%;ALP-OA/UA纳米颗粒可以显着抑制CuSO4引起的斑马鱼炎症反应,RT-PCR分析表明,这种抗炎活性可以通过下调NF-κB信号通路中的NF-κB2、TNF-α、IL-1β和IL-8来实现。
为了实现上述目的,本发明的技术方案如下所述:
在本发明的第一方面,提供一种复合抗炎纳米颗粒,所述纳米颗粒由熊果酸、齐墩果酸和牛蒡多糖组成,熊果酸和齐墩果酸被包覆在牛蒡多糖中;
其中,熊果酸和齐墩果酸的质量比为1~3:1~3;
熊果酸和齐墩果酸的质量的和与牛蒡多糖的质量比为1:10~100;
在本发明的第二方面,提供一种第一方面所述复合抗炎纳米颗粒的制备方法,包括以下步骤:
(1)从牛蒡中提取牛蒡多糖:将牛蒡粉碎,水提两次,上清液旋转蒸发浓缩后,进行脱脂和脱蛋白,纯化的牛蒡多糖用无水乙醇沉淀,冻干;
(2)制备牛蒡多糖-熊果酸-齐墩果酸复合抗炎纳米颗粒:
取步骤(1)得到的牛蒡多糖加水配制成溶液,取熊果酸和齐墩果酸用乙醇配制溶液;在牛蒡多糖溶液中加入熊果酸和齐墩果酸溶液,搅拌、旋蒸后离心,弃去未溶的熊果酸和齐墩果酸沉淀,取清液冻干即得。
在本发明的第三方面,提供一种上述复合抗炎纳米颗粒在制备抗炎药物中的应用。
在本发明的第四方面,提供一种抗炎药物,所述抗炎药物的活性成分包含上述复合抗炎纳米颗粒。
在本发明的第五方面,提供一种治疗与炎症相关疾病的方法,所述方法包括对受试者施用上述复合抗炎纳米颗粒和/或抗炎药物。
本发明的具体实施方式具有以下有益效果:
所获得的ALP-OA/UA纳米粒子能有效提高OA和UA的水溶性和分散性,包封率达到48.98%,负载量达到0.96%;ALP-OA/UA纳米颗粒的粒度为199.1nm,zeta电位为-7.15mV;ALP和ALP-OA/UA纳米颗粒可以显着抑制CuSO4引起的斑马鱼炎症反应。RT-PCR分析表明,这种抗炎活性可以通过下调NF-κB信号通路中的NF-κB2、TNF-α、IL-1β和IL-8来实现。这些结果表明OA和UA的抗炎活性具有协同作用,并且它们与ALP一起封装成纳米颗粒具有作为食品和药物制剂中疏水性生物活性分子的天然递送系统的潜力。
本发明中研究了熊果酸和齐墩果酸的抗炎活性,发现熊果酸和齐墩果酸的抗炎活性具有协同作用,并且本发明将牛蒡多糖(ALP)与熊果酸、齐墩果酸相结合,使用抗溶剂沉淀法制备了ALP-OA/UA纳米颗粒,所获得的ALP-OA/UA纳米粒子能有效提高OA和UA的水溶性和分散性,包封率达到48.98%,负载量达到0.96%;ALP-OA/UA纳米颗粒的粒度为199.1nm,zeta电位为-7.15mV;ALP-OA/UA纳米颗粒可以显着抑制CuSO4引起的斑马鱼炎症反应;RT-PCR分析表明,这种抗炎活性可以通过下调NF-κB信号通路中的NF-κB2、TNF-α、IL-1β和IL-8来实现。
本发明发现了OA和UA的抗炎活性具有协同作用,并且它们与ALP一起封装成纳米颗粒具有作为食品和药物制剂中疏水性生物活性分子的天然递送系统的潜力。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例1中浓度为1μg/mL不同比例OA/UA在斑马鱼模型上的抗炎效果;实验进行三次重复,数据表达为平均值±SD;*P<0.05,**P<0.01和***P<0.001与模型组相比;与对照组相比,####P<0.0001;
图2为本发明实施例1中不同比例OA/UA斑马鱼表形图;
图3为本发明实施例1中纳米颗粒包封率测定;
图4为本发明实施例1中牛蒡多糖及纳米颗粒扫描电镜与透射电镜图;
图5为本发明实施例1中牛蒡多糖及纳米颗粒的FTIR图;
图6为本发明实施例1中牛蒡多糖及纳米颗粒的XRD图;
图7为本发明实施例1中ALP和ALP-OA/UA抗炎结果图;
图8为本发明实施例1中牛蒡多糖及纳米颗粒斑马鱼表形图,实验进行三次重复,数据表达为平均值±SD;*P<0.05,**P<0.01和***P<0.001与模型组相比;与对照组相比,####P<0.0001;
图9为本发明实施例1中牛蒡多糖及纳米颗粒四种炎症因子表达;实验进行三次重复,数据表达为平均值±SD;*P<0.05和**P<0.01与模型组相比;与对照组相比,###P<0.001,##P<0.01;
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本申请提供进一步的说明。除非另有指明,本申请使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本发明的一种实施方式中,提供了一种复合抗炎纳米颗粒,所述纳米颗粒由熊果酸、齐墩果酸和牛蒡多糖组成,熊果酸和齐墩果酸被包覆在牛蒡多糖中;
其中,熊果酸和齐墩果酸的质量比为1~3:1~3,优选为1:1;
熊果酸和齐墩果酸的质量的和与牛蒡多糖的质量比为1:10~100;
本发明的一种实施方式中,提供了一种上述复合抗炎纳米颗粒的制备方法,包括以下步骤:
(1)从牛蒡中提取牛蒡多糖:将牛蒡粉碎,水提两次,上清液旋转蒸发浓缩后,进行脱脂和脱蛋白,纯化的牛蒡多糖用无水乙醇沉淀,冻干;
(2)制备牛蒡多糖-熊果酸-齐墩果酸复合抗炎纳米颗粒:
取步骤(1)得到的牛蒡多糖加水配制成溶液,取熊果酸和齐墩果酸用乙醇配制溶液;在牛蒡多糖溶液中加入熊果酸和齐墩果酸溶液,搅拌、旋蒸后离心,弃去未溶的熊果酸和齐墩果酸沉淀,取清液冻干即得。
在一种或多种实施方式中,步骤(1)中水提的温度为70-90℃,牛蒡和水的质量比为1:8-10;水提每次1.5-2小时;
在一种或多种实施方式中,步骤(1)中旋转蒸发的温度为50-70℃;
在一种或多种实施方式中,步骤(1)中脱脂和脱蛋白分别用石油醚和Sevag试剂(氯仿:丁醇=4:1,v/v)进行;
在一种或多种实施方式中,步骤(2)中熊果酸和齐墩果酸用70%乙醇配制溶液;
在一种或多种实施方式中,步骤(2)中向牛蒡多糖溶液中加入熊果酸和齐墩果酸溶液时,在300-400rpm磁力搅拌下将熊果酸和齐墩果酸溶液往牛蒡多糖中少量缓慢加入;
在一种或多种实施方式中,步骤(2)中旋蒸温度为40-44℃,离心10000-12000转;
本发明的一种实施方式中,提供了一种上述复合抗炎纳米颗粒在制备抗炎药物中的应用。
本发明的一种实施方式中,提供了一种抗炎药物,所述抗炎药物的活性成分包含上述复合抗炎纳米颗粒。
本发明的一种实施方式中,提供了一种治疗与炎症相关疾病的方法,所述方法包括对受试者施用上述复合抗炎纳米颗粒和/或抗炎药物。
下面结合具体的实施例对本发明作进一步的解释和说明。
牛蒡干切片,购于江苏沛县轶伟有限公司,品种为柳川理想牛蒡;成年斑马鱼于山东省科学院生物所获得(中国济南);齐墩果酸、熊果酸和布洛芬购于上海源叶有限公司;二甲基亚砜购于北京索莱宝科技有限公司;无水乙醇、石油醚、乙酸乙酯和正丁醇均购于天津凯通化学试剂有限公司;五水合硫酸铜(Ⅱ)(硫酸铜)购于国药集团化学试剂有限公司。
实施例1
齐墩果酸和熊果酸比例筛选
以转基因斑马鱼(Lyz:EGFP)为动物模型,在受精卵发育3dpf时,在体视显微镜下挑选正常的斑马鱼胚胎,移入24孔培养板中,每孔15枚。分别以OA:UA=1:0、3:1、1:1、1:3、0:1不同比例加入不同浓度样品的溶液(10-5-1μg/ml),加培养水至2mL。然后加盖,分别将受试组与阴性对照组斑马鱼置于光照培养箱(28℃)让胚胎继续发育。药物保护3h后用40μM的CuSO4分别处理以上各组斑马鱼,CuSO4避光处理斑马鱼1h后,观察。
数据分析报告了实验数据作为平均值±SE,使用ANOVA分析了显着的差异,P<0.05和P<0.01表示显着且非常显着差异。
CuSO4为引起斑马鱼急性炎症的物质,以巨噬细胞向神经的快速迁移为突出特征,加入抗炎药会导致这些巨噬细胞重新返回,因此可以使用此观察结果来评估UA和OA的抗炎活性。图2为不同比例的样品(1-1、1-2、1-3、1-4、1-5分别对应OA:UA=1:0、3:1、1:1、1:3、0:1)在1-5-10μg/ml不同浓度下斑马鱼抗炎效果图。由图1可知,硫酸铜组的巨噬细胞的数量明显多于空白对照组且有显著性差异,说明利用硫酸铜溶液诱导斑马鱼炎症模型是成功的。阳性对照组加入布洛芬后,巨噬细胞的数量明显减少,以此作为对照,发现不同比例的萜酸混合物均具有一定的抗炎作用,其中1-3即OA:UA=1:1时巨噬细胞数量较模型组(硫酸铜造模)下降29.55%-51.14%,抗炎效果最好。因此,选择1-3即OA:UA=1:1进行后续纳米颗粒制备以及活性评价实验。
实施例2
牛蒡多糖的提取、分离与纯化
从牛蒡中提取多糖:将干燥的牛蒡片在高速粉碎机中研磨,然后在80℃下水提取(10:1,v/w)两次,每次1.5小时;上清液用旋转蒸发仪60℃浓缩后,分别用石油醚和Sevag试剂(氯仿:丁醇=4:1,v/v)进行脱脂和脱蛋白;纯化的多糖用4倍体积的无水乙醇沉淀,离心后复溶蒸发乙醇后冻干备用;
牛蒡多糖-熊果酸-齐墩果酸纳米颗粒的制备
称取0.25g牛蒡多糖配制0.5%的牛蒡多糖水溶液50ml,称取熊果酸-齐墩果酸10mg(熊果酸和齐墩果酸5mg)用70%乙醇配制0.5mg/mL的熊果酸-齐墩果酸溶液10ml,均超声30min促进溶解,其中牛蒡多糖:熊果酸-齐墩果酸=50:1;取50mL的牛蒡多糖溶液,加入10mL的熊果酸-齐墩果酸溶液(300rpm磁力搅拌下将熊果酸-齐墩果酸溶液往糖溶液里加,少量缓慢加入),搅拌30min,42℃旋蒸后10000转离心,弃去未溶的萜酸沉淀,清液即为牛蒡多糖和熊果酸-齐墩果酸复合物溶液,冻干复溶后测定其中含量。
实施例3
牛蒡多糖的提取、分离与纯化
从牛蒡中提取多糖:将干燥的牛蒡片在高速粉碎机中研磨,然后在85℃下水提取(10:1,v/w)两次,每次2小时;上清液用旋转蒸发仪65℃浓缩后,分别用石油醚和Sevag试剂(氯仿:丁醇=4:1,v/v)进行脱脂和脱蛋白;纯化的多糖用4倍体积的无水乙醇沉淀,离心后复溶蒸发乙醇后冻干备用;
牛蒡多糖-熊果酸-齐墩果酸纳米颗粒的制备
称取0.2g牛蒡多糖配制2%的牛蒡多糖水溶液10ml,称取熊果酸-齐墩果酸10mg(熊果酸和齐墩果酸各5mg)用70%乙醇配制5mg/mL的熊果酸-齐墩果酸溶液2ml,均超声30min促进溶解,其中牛蒡多糖:熊果酸-齐墩果酸=20:1;取10mL的牛蒡多糖溶液,加入2mL的熊果酸-齐墩果酸溶液(300rpm磁力搅拌下将熊果酸-齐墩果酸溶液往糖溶液里加,少量缓慢加入),搅拌30min,42℃旋蒸后10000转离心,弃去未溶的萜酸沉淀,清液即为牛蒡多糖和熊果酸-齐墩果酸复合物溶液,冻干复溶后测定其中含量。
下面将以实施例2制备的牛蒡多糖-熊果酸-齐墩果酸纳米颗粒为试验对象进行测试:
纳米颗粒包封率及载药率测定
将适当浓度样品通过0.22μm微孔过滤器纯化用于HPLC分析;OA和UA由配备二极管阵列检测器的Shimadzu LC-20A高效液相色谱(HPLC)系统(Shimadzu,Kyoto,Japan)测定;使用Symmetry C18柱(Waters,4.6×250mm,5μm)在210nm波长下进行分析如下:0.2%的甲酸水溶液和甲醇作为流动相,柱温30℃,1.00mL/min的流速和10μL的进样体积。包封效率(EE)和负载能力(LC)由以下等式计算:
本发明比较了OA、UA、ALP和ALP-OA/UA在水中的分散性。从图3中可以明显的看出,齐墩果酸和熊果酸不溶于水;经过牛蒡多糖包埋后,OA和UA在水中分散性显著提高。且OA和UA与ALP自组装成纳米颗粒后共检测到9.604±0.74mg/g,表明OA和UA通过纳米技术获得了更高的水溶性,所得纳米颗粒的包封率为48.98±3.77%,负载量为0.96±0.07%。
采用衰减全反射-傅里叶红外光谱(FTIR-ATR)对ALP和ALP-OA/UA进行分析;如图5所示,4000-400cm-1范围内的吸收带为多糖的特征峰,其中3290cm-1处的峰属于O-H的伸缩振动。2930cm-1处的吸收峰属于C-H键的伸缩振动包括CH,CH2和CH3基团,1650cm-1和1420cm-1处的峰分别对应于对称的C=O伸缩振动和不对称的C=O的伸缩振动耦合C-H的弯曲振动。C-O-H和C-O-C的伸缩振动引起从1200cm-1到1000cm-1处的吸收。指纹区域1000-800cm-1处的吸收带为呋喃糖残留物的特征峰,其中934cm-1和818cm-1处的吸收峰分别对应于β-糖苷键和α-糖苷键。在ALP-OA/UA中找不到OA和UA的大多数特征峰,该结果表明UA和OA的特征峰与多糖的吸收带合并或重叠,表明OA和UA成功地包封到纳米颗粒中。由3290cm-1向3330cm-1的迁移可能是OA和UA与ALP之间形成氢键,氢键的形成是影响复合纳米粒子微观结构和性能的重要因素。
采用扫描电镜和透射电镜对ALP和ALP-OA/UA样品进行测试;结果见图4,其中Ⅰ是扫描倍数为5000的扫描电镜图,Ⅱ是透射电镜图,A为ALP-OA/UA的两种电镜图,B为ALP的两种电镜图;图B-Ⅰ显示了菊糖异构体的典型盘状形态。且从A-Ⅱ中也可看出,ALP-OA/UA在水中颗粒较小且呈疏松的网络状,从B-Ⅱ可看出ALP呈现明显的聚集现象,这可能是由于在干燥过程中聚合物链之间形成了牢固的氢键,另一个原因可能是ALP作为载体存在空间位阻效应,从而可以在一定程度上阻止粒子的聚集。
利用X-射线衍射分析仪对ALP和ALP-OA/UA冷冻干燥粉末的晶体性质进行研究,利用XRD谱图判断测定样品的晶体结构;得到的结果如图6所示,在2θ为21.67°、17.73°和12.00°处分别出现了明显的扩散峰,而ALP-OA/UA峰值明显降低,结晶度下降,表明包埋熊果酸-齐墩果酸后对ALP的结晶度有降低作用,可能是ALP与熊果酸-齐墩果酸之间的非共价作用,与红外结果一致。另外,已知UA和OA为晶体结构,由XRD可知ALP-OA/UA中未出现除多糖峰形以外的其他峰值,说明熊果酸-齐墩果酸与ALP结合后以无定形状态存在。据报道,无定形药物具有更好的水溶性和更快的溶解速度,这有助于提高该化合物在大多数食品和水性体系中的溶解度,进而提高OA及UA的生物利用度。
基于斑马鱼模型的ALP和ALP-OA/UA体内抗炎机制研究
以转基因斑马鱼(Lyz:EGFP)为动物模型,在受精卵发育3dpf时,在体视显微镜下挑选正常的斑马鱼胚胎,移入6孔培养板中,每孔30枚。分别加入不同浓度样品的溶液(10-100-200μg/ml),加培养水至5mL。然后加盖,分别将受试组与阴性对照组斑马鱼置于光照培养箱(28℃)让胚胎继续发育。药物保护3h后用40μM的CuSO4分别处理以上各组斑马鱼,CuSO4避光处理斑马鱼1h后,观察;
图7和图8为ALP和ALP-OA/UA抗炎结果图,由图7和图8可知,硫酸铜组的巨噬细胞的数量明显多于空白对照组且有显著性差异,说明利用硫酸铜溶液诱导斑马鱼炎症模型是成功的。阳性对照组加入布洛芬后,巨噬细胞的数量明显减少,以此作为对照,发现两种样品在不同浓度(10-100-200μg/mL)时,均具有一定的抗炎效果,但ALP-OA/UA组抗炎效果均高于ALP不同浓度组,且与模型组具有显著性差异,说明ALP通过包埋萜酸后提高了其抗炎效果。因此,为进一步研究其抗炎机制,通过PCR进行抗炎机制研究。
荧光定量PCR
经药物处理后的斑马鱼,用新鲜养鱼水冲洗两遍后吸干水,根据诺维赞试剂盒提取总RNA,将RNA逆转录以获得cDNA,使用RT-PCR检测系统测定炎症相关基因的表达水平。于实时定量PCR扩增仪反应器中设定反应条件:95℃,5min;1个循环后,95℃,10s,60℃,30s;40个循环后,95℃,15s,60℃,60s,95℃,15s;1个循环。以β-actin为内参,对目的基因的表达进行分析,用于RT-PCR的引物的序列如表1所示。
表1 引物的设计与合成
炎症细胞因子例如IL-1β,TNF-α和IL-8在炎症反应中发挥重要作用,TNF-α是一种免疫调节的细胞因子在炎症性疾病的发病过程中是关键的促炎因子,由特定的免疫细胞产生,能够激活NF-кB信号通路,上调P65和P50蛋白的表达量,促使促炎因子的分泌从而加快炎症反应的发生。IL-1β主要由先天性免疫细胞在炎症损伤中表达;IL-8可以激活嗜中性粒细胞,导致中性粒细胞产生一系列活性分子并导致炎症反应。通过测定促炎因子的mRNA表达量,结果显示空白组中炎症细胞因子的mRNA表达水平很低,而硫酸铜模型组中四种因子的mRNA表达量显著上升(见图9),且ALP和ALP-OA/UA不同浓度组四种因子的mRNA表达量显著下调,表明ALP和ALP-OA/UA可以通过抑制炎性细胞因子表达以减轻硫酸铜诱导的炎症。因此,推断两种物质均可能通过激活tnf-α和il-1β的表达进而调控nf-кB信号通路来达到抗炎效果。其中纳米颗粒的nf-кb2的表达量较多糖下降5.24%-31.15%,tnf-α的表达量较多糖下降15.64%-26.91%。结合表形中ALP-OA/UA的抗炎效果均好于ALP组进行分析,因此推测ALP与熊果酸-齐墩果酸结合后更有利于通过激活tnf-α进而调控nf-кB信号通路发挥作用。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。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)
1.一种复合抗炎纳米颗粒,其特征在于,所述纳米颗粒由熊果酸、齐墩果酸和牛蒡多糖组成,熊果酸和齐墩果酸被包覆在牛蒡多糖中;
其中,熊果酸和齐墩果酸的质量比为1~3:1~3;
熊果酸和齐墩果酸的质量的和与牛蒡多糖的质量比为1:10~100。
2.权利要求1所述复合抗炎纳米颗粒的制备方法,其特征在于,包括以下步骤:
(1)从牛蒡中提取牛蒡多糖:将牛蒡粉碎,水提两次,上清液旋转蒸发浓缩后,进行脱脂和脱蛋白,纯化的牛蒡多糖用无水乙醇沉淀,冻干;
(2)制备牛蒡多糖-熊果酸-齐墩果酸复合抗炎纳米颗粒:
取步骤(1)得到的牛蒡多糖加水配制成溶液,取熊果酸和齐墩果酸用乙醇配制溶液;在牛蒡多糖溶液中加入熊果酸和齐墩果酸溶液,搅拌、旋蒸后离心,弃去未溶的熊果酸和齐墩果酸沉淀,取清液冻干即得。
3.如权利要求2所述复合抗炎纳米颗粒的制备方法,其特征在于,步骤(1)中水提的温度为70-90℃,牛蒡和水的质量比为1:8-10;水提每次1.5-2小时。
4.如权利要求2所述复合抗炎纳米颗粒的制备方法,其特征在于,步骤(1)中旋转蒸发的温度为50-70℃。
5.如权利要求2所述复合抗炎纳米颗粒的制备方法,其特征在于,步骤(1)中脱脂和脱蛋白分别用石油醚和Sevag试剂进行。
6.如权利要求2所述复合抗炎纳米颗粒的制备方法,其特征在于,步骤(2)中向牛蒡多糖溶液中加入熊果酸和齐墩果酸溶液时,在300-400rpm磁力搅拌下将熊果酸和齐墩果酸溶液往牛蒡多糖中少量缓慢加入。
7.如权利要求2所述复合抗炎纳米颗粒的制备方法,其特征在于,步骤(2)中旋蒸温度为40-44℃,离心10000-12000转。
8.一种权利要求1所述的复合抗炎纳米颗粒在制备抗炎药物中的应用。
9.一种抗炎药物,其特征在于,所述抗炎药物的活性成分包含权利要求1所述的复合抗炎纳米颗粒。
10.一种治疗与炎症相关疾病的方法,其特征在于,所述方法包括对受试者施用权利要求1所述的纳米颗粒和/或权利要求9所述的抗炎药物。
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