红景天苷纳米脂质体的研制
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摘要
红景天中的主要活性成分红景天苷具有良好的保健作用,已经应用于医药、食品、化妆品等领域。但开发红景天苷的保健产品仍存在提取效率低、产品纯度低和体内生物利用度低等问题。
本文系统研究了红景天苷的提取分离纯化工艺;制备了红景天苷纳米脂质体,研究其物化稳定性;并进一步制备了红景天苷前体脂质体,研究其复水效果和贮藏稳定性;建立了红景天苷纳米脂质体的体外释放模型;研究了红景天苷纳米脂质体的抗辐射功能,探讨了红景天苷纳米脂质体在小鼠体内的组织分布和口服吸收动力学。
首先,采用微波辅助提取的方法提取红景天苷,结果表明微波功率为130 W,20%(v/v)乙醇作提取溶剂,固液比为1:29(g/mL),微波辐照时间60 s时,红景天苷的一次提取率达到80%。与传统的乙醇回流法相比,微波1 min提取两次和采用传统乙醇加热回流120min提取四次的结果基本相同。
采用大孔吸附树脂和超滤膜分离纯化红景天苷,结果表明DA201大孔吸附树脂对红景天苷有良好的吸附选择性,静态饱和吸附容量和动态吸附容量分别为21.88和13.25 mg/mL,通过柱层析实验确定了DA201树脂分离红景天苷的工艺,经50%乙醇一步洗脱可将红景天苷的纯度从3.8%提高到13.8%。超滤结果表明最佳超滤条件为压力0.05 MPa,温度40℃,提取液稀释倍数3倍,得到的红景天苷产品纯度较高,使用截留相对分子质量为10000和3000的超滤膜,红景天苷得率分别为86.2%和80.4%,红景天苷的纯度分别可达到30.65%和38.72%,满足工业化生产的需要。
采用薄膜法、冻融法、反相蒸发法、超声法、熔融法制备了红景天苷纳米脂质体。结果表明,红景天苷纳米脂质体的包封率依次为:冻融法＞薄膜法＞反相蒸发法＞熔融法＞超声法;红景天苷载量显著影响脂质体的包封率、粒径分布和zeta电位大小;超声法、熔融法和反相蒸发法制备的红景天苷纳米脂质体具有较好的分散性,PDI较低。红景天苷纳米脂质体的渗漏结果表明渗漏与时间的平方根呈线性关系,渗漏具有扩散特征。熔融法制备的红景天苷纳米脂质体显示较低的渗漏率,4℃和30℃下贮藏一个月的渗漏率分别为10%和15%。冻融法和薄膜法制备的红景天苷纳米脂质体在4℃和30℃下的物化稳定性最差,聚集现象严重;熔融法制备的红景天苷纳米脂质体4℃和30℃下经过180天的贮藏之后粒径能够保持基本不变。通过与空白脂质体的比较发现,红景天苷能够提高脂质体的稳定性。结果还表明红景天苷纳米脂质体的稳定性是空间稳定的结果。
采用乙醇注入法制备红景天苷纳米脂质体,结果显示红景天苷载量5%,胆固醇/磷脂质量比1:4,Tween80/磷脂摩尔比1:2,离子强度20-50 mmol/L,水合温度50℃,水合时间30 min,超声功率280W,制备的红景天苷纳米脂质体包封率较高,粒径小于100 nm,zeta电位为-10到-20 mV。体外释放结果表明,模拟胃液或肠液中24 h后,红景天苷累积释放率为60%,缓释效果明显。以红景天苷的包封率和一个月后的渗漏率为主要指标,进一步采取正交实验优化工艺条件。结果得到,胆固醇/磷脂比例为1:5,磷脂浓度1%,Tween80/磷脂摩尔比1:2,水合介质离子强度50 mmol/L的最佳工艺条件,制备的红景天苷纳米脂质体包封率为45.6%,一个月后的渗漏率为8.5%,粒径为147.6 nm,zeta电位为-12.5 mV。红景天苷纳米脂质体的物理稳定性结果表明,4℃和30℃条件下经过180天的贮藏之后,红景天苷纳米脂质体粒径分别增加21%和250%。AFM结果显示贮藏过程中红景天苷纳米脂质体粒径不断增大,形态变化。红景天苷纳米脂质体的化学稳定性研究结果表明,MDA含量持续上升和pH值持续下降,温度越高变化趋势越大。
通过实验筛选出甘露醇为最佳的冻干保护剂,成功制备红景天苷前体脂质体。甘露醇/磷脂质量比20:1,红景天苷前体脂质体外观饱满、致密,复水水合之后的包封率达到40%以上,包封率的保留率达到90%,粒径为223.9 nm,与红景天苷纳米脂质体粒径190.1 nm的结果十分接近。FTIR红外光谱分析显示磷脂极性头基和甘露醇的确发生了相互作用,形成氢键,起到了保护脂质体膜完整性的作用。红景天苷前体脂质体的贮藏稳定性结果表明,20℃或40℃条件下贮藏60天,外观、粒径、包封率、pH值、MDA等指标基本保持稳定。吸湿增重结果提示红景天苷前体脂质体贮藏过程中应尽量避免高湿。
针对红景天苷纳米脂质体体系,建立了简单、适用,能够描述红景天苷纳米脂质体释放性能的模型。模型预示了红景天苷分子在起始阶段具有突释现象,综合分析了突释同胆固醇和Tween80用量,以及红景天苷载量等制备因素之间的关系。对实验数据进行了模型参数的拟合,通过不同的释放速率和模型参数的比较,揭示了胆固醇和Tween80用量,以及红景天苷载量等制备因素同释放性能之间的一些基本规律。
小鼠X射线亚急性和亚慢性辐射损伤防护实验表明,红景天苷纳米脂质体对小鼠亚急性和亚慢性辐射损伤具有良好的防护作用。小鼠体内组织分布结果显示,红景天苷纳米脂质体对组织的亲和力增强,改变了在组织中的分布,对肝、脑等特定的组织具有一定的富集效果。药代动力学结果表明,红景天苷纳米脂质体相对于红景天苷提取液在体内有较长的滞后时间和较低的清除率,释放更为缓慢,延长了有效作用时间。因此,红景天苷纳米脂质体的生物利用度得到了增加,纳米脂质体是一种能够提高红景天苷生物有效性的优良载体。
The major bioactive components in Rhodiola species is salidroside.Salidroside has been shown to possess the functions such as resisting anoxia,fatigue and radiation.It's reported to be widely used in medicine,food and cosmetic.Unfortunately,functional foods containing salidroside presently developed have some drawbacks,such as low extraction efficiency,low purity and low bioavailability.
Separation and purification of salidroside was operated systemically.Salidroside nano-liposomes were prepared by different methods and the physicochenmical properties of salidroside nano-liposomes were investigated.Furthermore,salidroside pro-liposomes were prepared by vacuum freeze-drying and rehydration and stability was investigated.A new mechanistic mathematical model for salidroside release from liposomes was proposed.Radioprotection of salidroside nano-liposomes was studied and in vivo tissue distribution and pharmacokinetic parameters of salidroside extract and salidroside nano-liposomes were investigated after oral administration.
Microwave-assisted extraction was employed to separate salidroside from Rhodiola species. Salidroside was obtained in a yield of 80%under the following condition:microwave powder,130 W; ethanol concentration,20%(v/v);solid/liquid ratio,1:29(g/mL),irradiation time,60 s.MAE gives almost 80%extraction efficiency at 1 min,whereas traditional heating extraction method gives about 60% extraction efficiency at 120 min.Ethanol used in conventional reflux is second higher than one used in MAE.
Results showed DA201 displayed the optimal adsorption and desorption properties.The static and dynamic adsorption capacities of DA201 were 21.88 and 13.25 mg/mL,respectively.The purity of the partially purified salidroside product was improved from 3.8%to 13.8%by one step separation and purification with 50%ethanol.Further purification by ultra-filtration was used to achieve the optimization: operation pressure was 0.05 MPa,temperature was 40℃and extract was diluted 3 times.Through cut-off MW 10000 and 3000,salidroside purity was 30.65%and 38.72%respectively and salidroside retention ratio was 86.2%and 80.4%,respectively.
Salidroside nano-liposomes were prepared by using five different methods:thin film evaporation, sonication,reverse phase evaporation,melting,and freezing-thawing.Results showed the encapsulating efficiency of liposomes was the following:freezing-thawing＞thin film evaporation＞reverse phase evaporation＞melting and sonieation.Loading capacity of salidroside had significant effect on encapsulating efficiency,average diameter and zeta potential of nano-liposomes.Liposomal systems prepared by sonication,melting and reverse phase evaporation displayed better dispersivity.Determination of leakage of salidroside from different liposomal systems revealed that melting method had the lowest leakage of 10%and 15%,at 4 and 30℃after one month of storage,respectively.In all cases,a straight-line leakage behavior of salidroside was found.This revealed that the leakage of salidroside was a diffusion process from the membrane of nano-liposomes.Furthermore,the storage stability of different liposomal systems showed that salidroside liposomes prepared by melting had a better physicochemical stability.Salidroside nano-liposomes showed the slower increase in particle size than liposomes without salidroside.Stability of salidroside nano-liposomes depended on dimensional structure.
Salidroside nano-liposomes were prepared by ethanol injection method.Higher encapsulating efficiency of salidroside was obtained with cholesterol to lipid mass ratio of 1:4,Tween 80 and lipid to the molar ratio of 1:2,and ion strength in a range 20-50 mmol/L.The particles of nano-liposomes were below 100 nm and zeta potential was in the range of-10 and -20 mV.The release study of salidroside in vitro from nano-liposomes exhibited a prolonged release profile as studied over a period of 24 h.Based on the above experiments,optimizations were investigated according to encapsulating efficiency and leakage ratio after one month of salidroside.Results showed optimizations were obtained with cholesterol to lipid mass ratio of 1:5,Tween 80 and lipid to the molar ratio of 1:2,and ion strength was 50 mmol/L.And encapsulating efficiency was 45.6%,leakage ratio after one month was 8.5%,particle size was 147.6 nm,zeta potential was -12.5 mV.Particle size of salidroside nano-liposomes was increased 21%and 250%after 180 days at 4℃and 30℃,respectively.AFM scanning image showed particle size of salidroside nano-liposomes increased constantly during the storage.MDA content increased and pH value decreased constantly. Instability was exaggerated with a rise in temperature.
Mannitol was used as the optimal cryprotectant to prepare salidroside pro-liposomes.Salidroside pro-liposomes showed full and compact appearance.After rehydration,encapsulating efficiency reached above 40%and retention ratio was above 90%.Compared with salidroside nano-liposomes of 190.1 nm before freezing-drying,particle size was 223.9 nm after rehydration.According to FTIR spectra,mannitol and polar group of lipid interacted and created hydrogen bonds,which could protect the bilayer of liposomes from being destroyed during freezing-drying.There happened almost change in appearance, particle size,encapsulating efficiency,pH value and MDA content of salidroside pro-liposomes in the condition of 20 or 40℃,stored 60 days.Results showed stability of salidroside pro-liposomes was satisfied. Results on moisture absorption experiments illuminated salidroside pro-liposomes must be avoided the higher relatively humidity condition during storage.
The mechanistic mathematical model was developed here for quantitative description of the release characteristics.Results showed the model gave excellent correlative accuracy within the release period.The model parameters,diffusion resistant,were theoretical sound and showed good trend,which could be correlated to various factors such as cholesterol content,Tween80 and salidroside loading capacity.The model investigated the effect of various factors on burst release profiles of salidroside based on limited experimental data.The model parameters can be used to describe the underlying mechanisms of the certain salidroside release process as well as for salidroside delivery device design and process optimization.These results suggested the new mechanistic mathematical model was practical for in vitro release of salidroside from liposomes.
Experimental results on sub-acute or sub-chronic X-radiation injured mice showed salidroside nano-liposomes had excellent radioprotection effect on sub-acute or sub-chronic X-radiation.In vivo tissue distribution of salidroside was different after oral salidroside nano-liposomes and salidroside extract. Salidroside concentration in liver and brain after oral salidroside nano-liposomes was higher than that of salidroside extract.Pharmacokinetics showed salidroside nano-liposomes had slower release rate,lower clearance rate and was delayed circulation time than salidroside extract.The bioavailability of salidroside was been improved greatly.Nano-liposomes can be used as an excellent carrier for salidroside.
本文系统研究了红景天苷的提取分离纯化工艺;制备了红景天苷纳米脂质体,研究其物化稳定性;并进一步制备了红景天苷前体脂质体,研究其复水效果和贮藏稳定性;建立了红景天苷纳米脂质体的体外释放模型;研究了红景天苷纳米脂质体的抗辐射功能,探讨了红景天苷纳米脂质体在小鼠体内的组织分布和口服吸收动力学。
首先,采用微波辅助提取的方法提取红景天苷,结果表明微波功率为130 W,20%(v/v)乙醇作提取溶剂,固液比为1:29(g/mL),微波辐照时间60 s时,红景天苷的一次提取率达到80%。与传统的乙醇回流法相比,微波1 min提取两次和采用传统乙醇加热回流120min提取四次的结果基本相同。
采用大孔吸附树脂和超滤膜分离纯化红景天苷,结果表明DA201大孔吸附树脂对红景天苷有良好的吸附选择性,静态饱和吸附容量和动态吸附容量分别为21.88和13.25 mg/mL,通过柱层析实验确定了DA201树脂分离红景天苷的工艺,经50%乙醇一步洗脱可将红景天苷的纯度从3.8%提高到13.8%。超滤结果表明最佳超滤条件为压力0.05 MPa,温度40℃,提取液稀释倍数3倍,得到的红景天苷产品纯度较高,使用截留相对分子质量为10000和3000的超滤膜,红景天苷得率分别为86.2%和80.4%,红景天苷的纯度分别可达到30.65%和38.72%,满足工业化生产的需要。
采用薄膜法、冻融法、反相蒸发法、超声法、熔融法制备了红景天苷纳米脂质体。结果表明,红景天苷纳米脂质体的包封率依次为:冻融法＞薄膜法＞反相蒸发法＞熔融法＞超声法;红景天苷载量显著影响脂质体的包封率、粒径分布和zeta电位大小;超声法、熔融法和反相蒸发法制备的红景天苷纳米脂质体具有较好的分散性,PDI较低。红景天苷纳米脂质体的渗漏结果表明渗漏与时间的平方根呈线性关系,渗漏具有扩散特征。熔融法制备的红景天苷纳米脂质体显示较低的渗漏率,4℃和30℃下贮藏一个月的渗漏率分别为10%和15%。冻融法和薄膜法制备的红景天苷纳米脂质体在4℃和30℃下的物化稳定性最差,聚集现象严重;熔融法制备的红景天苷纳米脂质体4℃和30℃下经过180天的贮藏之后粒径能够保持基本不变。通过与空白脂质体的比较发现,红景天苷能够提高脂质体的稳定性。结果还表明红景天苷纳米脂质体的稳定性是空间稳定的结果。
采用乙醇注入法制备红景天苷纳米脂质体,结果显示红景天苷载量5%,胆固醇/磷脂质量比1:4,Tween80/磷脂摩尔比1:2,离子强度20-50 mmol/L,水合温度50℃,水合时间30 min,超声功率280W,制备的红景天苷纳米脂质体包封率较高,粒径小于100 nm,zeta电位为-10到-20 mV。体外释放结果表明,模拟胃液或肠液中24 h后,红景天苷累积释放率为60%,缓释效果明显。以红景天苷的包封率和一个月后的渗漏率为主要指标,进一步采取正交实验优化工艺条件。结果得到,胆固醇/磷脂比例为1:5,磷脂浓度1%,Tween80/磷脂摩尔比1:2,水合介质离子强度50 mmol/L的最佳工艺条件,制备的红景天苷纳米脂质体包封率为45.6%,一个月后的渗漏率为8.5%,粒径为147.6 nm,zeta电位为-12.5 mV。红景天苷纳米脂质体的物理稳定性结果表明,4℃和30℃条件下经过180天的贮藏之后,红景天苷纳米脂质体粒径分别增加21%和250%。AFM结果显示贮藏过程中红景天苷纳米脂质体粒径不断增大,形态变化。红景天苷纳米脂质体的化学稳定性研究结果表明,MDA含量持续上升和pH值持续下降,温度越高变化趋势越大。
通过实验筛选出甘露醇为最佳的冻干保护剂,成功制备红景天苷前体脂质体。甘露醇/磷脂质量比20:1,红景天苷前体脂质体外观饱满、致密,复水水合之后的包封率达到40%以上,包封率的保留率达到90%,粒径为223.9 nm,与红景天苷纳米脂质体粒径190.1 nm的结果十分接近。FTIR红外光谱分析显示磷脂极性头基和甘露醇的确发生了相互作用,形成氢键,起到了保护脂质体膜完整性的作用。红景天苷前体脂质体的贮藏稳定性结果表明,20℃或40℃条件下贮藏60天,外观、粒径、包封率、pH值、MDA等指标基本保持稳定。吸湿增重结果提示红景天苷前体脂质体贮藏过程中应尽量避免高湿。
针对红景天苷纳米脂质体体系,建立了简单、适用,能够描述红景天苷纳米脂质体释放性能的模型。模型预示了红景天苷分子在起始阶段具有突释现象,综合分析了突释同胆固醇和Tween80用量,以及红景天苷载量等制备因素之间的关系。对实验数据进行了模型参数的拟合,通过不同的释放速率和模型参数的比较,揭示了胆固醇和Tween80用量,以及红景天苷载量等制备因素同释放性能之间的一些基本规律。
小鼠X射线亚急性和亚慢性辐射损伤防护实验表明,红景天苷纳米脂质体对小鼠亚急性和亚慢性辐射损伤具有良好的防护作用。小鼠体内组织分布结果显示,红景天苷纳米脂质体对组织的亲和力增强,改变了在组织中的分布,对肝、脑等特定的组织具有一定的富集效果。药代动力学结果表明,红景天苷纳米脂质体相对于红景天苷提取液在体内有较长的滞后时间和较低的清除率,释放更为缓慢,延长了有效作用时间。因此,红景天苷纳米脂质体的生物利用度得到了增加,纳米脂质体是一种能够提高红景天苷生物有效性的优良载体。
The major bioactive components in Rhodiola species is salidroside.Salidroside has been shown to possess the functions such as resisting anoxia,fatigue and radiation.It's reported to be widely used in medicine,food and cosmetic.Unfortunately,functional foods containing salidroside presently developed have some drawbacks,such as low extraction efficiency,low purity and low bioavailability.
Separation and purification of salidroside was operated systemically.Salidroside nano-liposomes were prepared by different methods and the physicochenmical properties of salidroside nano-liposomes were investigated.Furthermore,salidroside pro-liposomes were prepared by vacuum freeze-drying and rehydration and stability was investigated.A new mechanistic mathematical model for salidroside release from liposomes was proposed.Radioprotection of salidroside nano-liposomes was studied and in vivo tissue distribution and pharmacokinetic parameters of salidroside extract and salidroside nano-liposomes were investigated after oral administration.
Microwave-assisted extraction was employed to separate salidroside from Rhodiola species. Salidroside was obtained in a yield of 80%under the following condition:microwave powder,130 W; ethanol concentration,20%(v/v);solid/liquid ratio,1:29(g/mL),irradiation time,60 s.MAE gives almost 80%extraction efficiency at 1 min,whereas traditional heating extraction method gives about 60% extraction efficiency at 120 min.Ethanol used in conventional reflux is second higher than one used in MAE.
Results showed DA201 displayed the optimal adsorption and desorption properties.The static and dynamic adsorption capacities of DA201 were 21.88 and 13.25 mg/mL,respectively.The purity of the partially purified salidroside product was improved from 3.8%to 13.8%by one step separation and purification with 50%ethanol.Further purification by ultra-filtration was used to achieve the optimization: operation pressure was 0.05 MPa,temperature was 40℃and extract was diluted 3 times.Through cut-off MW 10000 and 3000,salidroside purity was 30.65%and 38.72%respectively and salidroside retention ratio was 86.2%and 80.4%,respectively.
Salidroside nano-liposomes were prepared by using five different methods:thin film evaporation, sonication,reverse phase evaporation,melting,and freezing-thawing.Results showed the encapsulating efficiency of liposomes was the following:freezing-thawing＞thin film evaporation＞reverse phase evaporation＞melting and sonieation.Loading capacity of salidroside had significant effect on encapsulating efficiency,average diameter and zeta potential of nano-liposomes.Liposomal systems prepared by sonication,melting and reverse phase evaporation displayed better dispersivity.Determination of leakage of salidroside from different liposomal systems revealed that melting method had the lowest leakage of 10%and 15%,at 4 and 30℃after one month of storage,respectively.In all cases,a straight-line leakage behavior of salidroside was found.This revealed that the leakage of salidroside was a diffusion process from the membrane of nano-liposomes.Furthermore,the storage stability of different liposomal systems showed that salidroside liposomes prepared by melting had a better physicochemical stability.Salidroside nano-liposomes showed the slower increase in particle size than liposomes without salidroside.Stability of salidroside nano-liposomes depended on dimensional structure.
Salidroside nano-liposomes were prepared by ethanol injection method.Higher encapsulating efficiency of salidroside was obtained with cholesterol to lipid mass ratio of 1:4,Tween 80 and lipid to the molar ratio of 1:2,and ion strength in a range 20-50 mmol/L.The particles of nano-liposomes were below 100 nm and zeta potential was in the range of-10 and -20 mV.The release study of salidroside in vitro from nano-liposomes exhibited a prolonged release profile as studied over a period of 24 h.Based on the above experiments,optimizations were investigated according to encapsulating efficiency and leakage ratio after one month of salidroside.Results showed optimizations were obtained with cholesterol to lipid mass ratio of 1:5,Tween 80 and lipid to the molar ratio of 1:2,and ion strength was 50 mmol/L.And encapsulating efficiency was 45.6%,leakage ratio after one month was 8.5%,particle size was 147.6 nm,zeta potential was -12.5 mV.Particle size of salidroside nano-liposomes was increased 21%and 250%after 180 days at 4℃and 30℃,respectively.AFM scanning image showed particle size of salidroside nano-liposomes increased constantly during the storage.MDA content increased and pH value decreased constantly. Instability was exaggerated with a rise in temperature.
Mannitol was used as the optimal cryprotectant to prepare salidroside pro-liposomes.Salidroside pro-liposomes showed full and compact appearance.After rehydration,encapsulating efficiency reached above 40%and retention ratio was above 90%.Compared with salidroside nano-liposomes of 190.1 nm before freezing-drying,particle size was 223.9 nm after rehydration.According to FTIR spectra,mannitol and polar group of lipid interacted and created hydrogen bonds,which could protect the bilayer of liposomes from being destroyed during freezing-drying.There happened almost change in appearance, particle size,encapsulating efficiency,pH value and MDA content of salidroside pro-liposomes in the condition of 20 or 40℃,stored 60 days.Results showed stability of salidroside pro-liposomes was satisfied. Results on moisture absorption experiments illuminated salidroside pro-liposomes must be avoided the higher relatively humidity condition during storage.
The mechanistic mathematical model was developed here for quantitative description of the release characteristics.Results showed the model gave excellent correlative accuracy within the release period.The model parameters,diffusion resistant,were theoretical sound and showed good trend,which could be correlated to various factors such as cholesterol content,Tween80 and salidroside loading capacity.The model investigated the effect of various factors on burst release profiles of salidroside based on limited experimental data.The model parameters can be used to describe the underlying mechanisms of the certain salidroside release process as well as for salidroside delivery device design and process optimization.These results suggested the new mechanistic mathematical model was practical for in vitro release of salidroside from liposomes.
Experimental results on sub-acute or sub-chronic X-radiation injured mice showed salidroside nano-liposomes had excellent radioprotection effect on sub-acute or sub-chronic X-radiation.In vivo tissue distribution of salidroside was different after oral salidroside nano-liposomes and salidroside extract. Salidroside concentration in liver and brain after oral salidroside nano-liposomes was higher than that of salidroside extract.Pharmacokinetics showed salidroside nano-liposomes had slower release rate,lower clearance rate and was delayed circulation time than salidroside extract.The bioavailability of salidroside was been improved greatly.Nano-liposomes can be used as an excellent carrier for salidroside.
引文
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