| Astaxanthin(AST)is a hydrophobic carotenoid with strong antioxidant capacity,which has been proved to have neuroprotective effects.However,due to its low water solubility and poor stability,it has low bioavailability in vivo.Using protein to prepare AST delivery system can improve its stability and biological activity.Studies have shown that Lactoferrin(LF)can cross the Blood brain barrier(BBB)through the transcellular mechanism of mouse brain capillary endothelial cells(BCECs).Meanwhile,there are a large number of LF specific receptors in the brain,so the construction of AST delivery system with LF as the carrier using the above characteristics can effectively exert the neuroprotective activity of AST.In this project,we first induced the thermal denaturation of lactoferrin by heating treatment to form nanoparticles.Then,we used the electrostatic self-assembly method,and took the cationic lactoferrin as the inner layer,and the anion polysaccharide(beet pectin,BP or carboxymethyl chitosan,CMCS)as the outer layer.The astaxanthin was coated in the double emulsion,and the physical and chemical properties and functional properties of the emulsion were systematically studied.The main contents are as follows:(1)Lactoferrin was modified by changing the temperature of heat treatment.A single layer and double layer emulsion of astaxanthin was constructed by using lactoferrin and polysaccharide as wall materials.The effects of thermal denaturation and addition of polysaccharides on the properties of the emulsion were explored.Circular dichroism spectra showed that the increase of thermal denaturation temperature led to a decrease in the ratio of the α-helical conformation in the secondary structure of lactoferrin,and an increase in the conformation of the β-fold.The AST monolayer emulsion prepared by high temperature thermal denaturation of LF had the smallest particle size and higher zeta potential level,and 95°C LF had the longest half-life and the highest emulsion stability in the light stability.The emulsion formed by LF had the smallest change in particle size and zeta potential during storage and showed good storage stability.In the aspect of micromorphology,LF stable monolayer emulsion can form evenly dispersed emulsion droplets.After adding anionic polysaccharides(BP or CMCS),LF-BP and LF-CMCS stabilized bilayer emulsion can be formed.Compared with CMCS,the double layer emulsion formed by BP has higher stability,better storage stability and stable astaxanthin effect.The activity of astaxanthin emulsion embedded at 95°C LF-BP was studied.(2)Taking the optimal emulsion at 95°C LF-AST-BP as the model drug,the metabolic distribution of this emulsion in rats and the accumulation of targeting to the brain were investigated.The method for the determination of astaxanthin in biological samples by UHPLC was successfully established and verified.Meanwhile,the pharmacokinetic test showed that the blood concentration of astaxanthin in rats increased with the change of time and reached the peak at 8 h.In tissue sample detection,the results showed that astaxanthin content in the brain of rats treated with astaxanthin emulsion was consistently higher than that in the natural astaxanthin group during 7～9 h after intragastric administration,which indicated that astaxanthin emulsion delivery system constructed with lactoferrin as carrier could effectively cross the blood-brain barrier and reach the brain for effective enrichment.(3)Interventional effects of 95°C LF-AST-BP emulsion on neuroinflammatory response induced by lipopolysaccharide(LPS)in C57BL6/J mice were studied.Through animal behavior experiments,it was found that dietary astaxanthin emulsion could effectively improve the cognitive and learning and memory impairment caused by inflammation in mice.The m RNA and protein immunoblotting results also showed that dietary astaxanthin emulsion could effectively inhibit the expression of inflammatory marker proteins in the brain of mice,such as i NOS,COX-2,IL-1β and TNF-α.Moreover,the expression of inflammatory marker genes,such as i NOS,COX-2,IL-1β,TNF-α and IL-6,were significantly inhibited.In addition,dietary astaxanthin supplementation increased the expression of neurotrophic factor-related genes in the brain of inflammatory mice,and to some extent increased the length and width of postsynaptic dense protein related to learning and memory in the brain of mice.(4)The effects of 95°C LF-AST-BP emulsion on the inflammation of BV2 neuromicroglia induced by LPS were investigated.The results indicated that astaxanthin emulsion could inhibit the expression of inflammatory marker proteins notably,such as i NOS,COX-2,IL-1β,TNF-α and IL-6,and significantly inhibit the expression of inflammatory marker genes,such as i NOS,COX-2,IL-1β,TNF-α and IL-6 induced by LPS.In addition,the intervention of astaxanthin emulsion significantly alleviated the decrease of mitochondrial membrane potential,inhibited the intracellular ATPase and NO levels,and improved mitochondrial function.In addition,the level of intracellular reducing GSH was significantly increased,intracellular ROS production was significantly inhibited,and intracellular redox balance was maintained.In conclusion,in this study,the thermal denatured lactoferrin was used as raw material,astaxanthin was used as loading material,and the layer-by-layer electrostatic self-assembly system was constructed by adding polysaccharides,so that the system had good stability and protected astaxanthin from oxidation.At the same time,through the in vivo experiment,we proved that the lactoferrin astaxanthin emulsion had good brain targeting effect and intervention effect on neuroinflammation in inflammatory mice.The intervention effect of astaxanthin emulsion on inflammatory reaction of microglia was verified by in vitro experiments,which provided a feasible strategy for the processing and utilization of natural astaxanthin,making it suitable for the development of food and health products. |
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