Effect Of Phytosterol Esters On The Properties And Membrane Structure Of Astaxanthin-loaded Liposomes

Astaxanthin is a kind of natural carotenoid with anti-cancer,anti-inflammatory,antioxidant and other physiological functions.However,astaxanthin has poor water solubility and poor stability,which greatly limits its application.Liposomes are spherical vesicles composed of amphiphilic phospholipids molecules with structure similar to cell membranes,which can incorporate hydrophilic,lipophilic or amphiphilic substances and have sustained release regulation.When preparing liposomes,cholesterol is usually added to achieve the purpose of stabilizing liposomes.While,high cholesterol levels will cause some health problems.Phytosterols have a variety of physiological functions beneficial to human health and similar molecular structure to cholesterol,which can replace cholesterol to stabilize liposome membranes.Nevertheless,the high melting point and low solubility limit the application of phytosterols.Phytosterol esters are obtained by esterification of phytosterols and fatty acids,which effectively improve the fat solubility,reduce melting point,and have excellent physicochemical properties and physiological functions.It is of great significance to apply phytosterol esters in the preparation of liposomes loaded with lipid soluble bioactive substances.In this paper,four kinds of liposomes co-encapsulating astaxanthin and phytosterol esters(phytosterol oleate ester,phytosterol linoleate ester,phytosterol ɑ-linolenate ester and phytosterol stearate ester)were prepared by thin-film ultrasonic method with soybean phospholipid as the main membrane material.This study optimized the preparation process of complex liposomes,and explored the effects of the four kinds of phytosterol esters with different structures on the properties and membrane structure characteristics of astaxanthin-loaded liposomes.The results were as follows:(1)Liposomes co-encapsulating astaxanthin and phytosterol oleate ester(L-A-POE)were prepared by thin-film ultrasonic method.The preparation of L-A-POE was optimized by single factor test and Box-Behnken response surface test with the encapsulation efficiency as the index.The optimal technological conditions obtained were as follows: the mass ratio of phytosterol oleate ester to soybean phospholipid was 1:10,the pH of phosphate buffer was 7.4,the ultrasonic treatment time was 4 min,the mass ratio of Tween 80 to soybean phospholipid was 1:2,the volume ratio of organic phase to aqueous phase was 1:4,and the mass ratio of astaxanthin to soybean phospholipid was 1:50.Under the optimum conditions,the encapsulation efficiency of L-A-POE was 95.24±0.45%,the particle size was156.6±3.3 nm,the polydispersity index was 0.360±0.01,and the zeta potential was-31.4±1.2m V.Transmission electron microscopy revealed that the complex liposomes were spherical with regular shape and uniform particle size distribution.(2)Compared with astaxanthin-loaded liposomes without phytosterol ester incorporated,the effects of phytosterol esters with different structures on the storage stability,heat treatment stability,light stability,pH stability and salt stability of astaxanthin-loaded liposomes were investigated by means of retention rate,particle size distribution and zeta potential.The results showed that the incorporation of phytosterol esters increased the retention rate of astaxanthin-loaded liposomes,which may be due to the synergistic antioxidant effect of phytosterol esters and astaxanthin,which slowed down the oxidative decomposition of astaxanthin to a certain extent.Among them,the particle size distribution of liposomes incorporated with phytosterol oleate ester,phytosterol linoleate ester and phytosterol ɑ-linolenate ester changed little under different treatment conditions,which improved the stability of astaxanthin-loaded liposomes.The different effects of phytosterol esters on the stability of astaxanthin-loaded liposomes were mainly due to the different saturation of phytosterol esters.The incorporation of phytosterol unsaturated fatty acid esters improved the stability of astaxanthin-loaded liposomes.While,the particle size distribution of astaxanthin-loaded liposomes incorporated with phytosterol stearate showed the greatest change and the worst uniformity,which decreased the stability.(3)Pyrene,ANS,DPH fluorescence probe and Raman spectroscopy were used to study the effect of phytosterol esters with different structures on the membrane fluidity and structural properties of astaxanthin-loaded liposomes bilayer.The results showed that the incorporation of phytosterol oleate ester,phytosterol linoleate ester and phytosterolɑ-linolenate ester improved the membrane properties of astaxanthin-loaded liposomes and improved the stability of the membrane structure of astaxanthin-loaded liposomes,while the incorporation of phytosterol stearate ester reduced the stability of astaxanthin-loaded liposomes.L-A-POE had the smallest membrane micropolarity and fluidity,the highest membrane surface structure stability,hydrophobicity and orderliness,and the highest fluorescence anisotropy.Liposomes incorporated with phytosterol linoleate ester and phytosterol ɑ-linolenate ester were the second.While,the incorporation of phytosterol stearate ester did not significantly improve the membrane properties of astaxanthin-loaded liposomes.he results of fluorescence lifetime and Raman spectroscopy also showed that the incorporation of phytosterol unsaturated fatty acid ester improved the stability of liposome membrane structure,while phytosterol stearate ester reduced the stability of astaxanthin-loaded liposomes.In conclusion,it is feasible to use phytosterol esters in the construction of astaxanthin-loaded liposomes.The incorporation of phytosterol unsaturated fatty acid ester could improve the stability and membrane properties of astaxanthin-loaded liposomes,while the incorporation of phytosterol stearate ester reduced the stability of astaxanthin-loaded liposomes.