Research On The Fabrication,Characterization,and Delivery Effect Of Salidroside Water-in-oil-in-water Double Emulsion

Phenolic compounds are secondary metabolites of plants and have important functional activities.Salidroside is a water-soluble natural phenolic compound from Rhodiola rosea.It has a variety of biological activities and has outstanding antioxidative,anti-inflammatory and anticancer effects.However,its application in food is severely limited by its short biological half-life and sensitivity to temperature,p H and other environmental factors.Water-in-oil-in-water（W/O/W）emulsions can be used as delivery carriers for functional raw food materials and protect water-soluble compounds in the internal water phase during digestion,and they have great potential in the development of healthy food.The aim of this study was to construct a salidroside W/O/W double emulsion using whey protein isolate（WPI）,sodium alginate（SA）and polyglycerol polyricinoleate（PGPR）as emulsifiers to investigate the effect and mechanism of salidroside W/O/W double emulsion on dextran sulphate sodium salt（DSS）-induced colitis and tetrachloromethane（CCl₄）-induced acute liver injury in mice.（1）Salidroside W/O/W double emulsions were prepared by a two-step emulsification method,and the effects of SA and WPI on the properties of salidroside W/O/W double emulsions were studied.The average particle size,particle size distribution,encapsulation efficiency,infrared spectroscopy,structure formation and microstructure of the salidroside W/O/W double emulsion were analyzed.The results showed that the characteristic peak of SA disappeared at 2919 cm^-1 and blue-shifted at1620 cm^-1 after emulsion formation.At the same time,the characteristic peaks of salidroside disappeared at 1259 and 1074 cm^-1,indicating that salidroside was successfully encapsulated in the emulsion system.The particle size of the salidroside W/O/W double emulsion was negatively correlated with the SA and WPI concentrations.In the range of 0%-5%,the encapsulation efficiency of salidroside first increased and then decreased.Combined with 2%SA,the encapsulation efficiency of salidroside in a W/O/W double emulsion of 4%WPI was 77.9±1.3%.Structurally,salidroside is in the inner water phase of the W/O/W double emulsion,and the oil droplets are embedded into the network structure of the outer water phase,the embedding degree is related to the concentration of WPI and SA.The combination of 4%WPI and 2%SA promoted the formation and densification of the network structure,protected the oil droplets from coalescence and improved the stability of the emulsion.（2）The effects of WPI and SA concentrations on the rheological properties and water distribution of salidroside W/O/W double emulsion were studied,combined with the results of storage stability,the optimal WPI and SA concentrations were determined.The optimal concentrations of WPI and SA were determined according to the storage stability of the salidroside W/O/W double emulsion.At this concentration,the environmental stability,antioxidant activity and in vitro simulated digestive property of the emulsion were continuously evaluated.The results showed that SA and WPI could promote the structural rearrangement of the salidroside W/O/W double emulsion system,enhance the viscosity characteristics of the emulsion and the ability to capture water molecules,which is conducive to the stability of the salidroside W/O/W double emulsion.The salidroside W/O/W double emulsion prepared at 2%SA and 4%WPI remained stable at 4°C for 28 days,the W/O/W double emulsion enhanced the protection of salidroside,and the free radical scavenging efficiency of the W/O/W double emulsion was improved.The salidroside W/O/W double emulsion effectively controlled the release of salidroside in a simulated gastrointestinal environment,and the release rate was only 47.9%after simulated digestion for 3 h.（3）The effect of salidroside W/O/W double emulsion on DSS induced colitis in mice was studied.Transcriptomic analysis and the determination of oxidative damage,inflammatory factors and gut microbiota were used to analyze the mechanism of alleviation.The results showed that salidroside W/O/W double emulsion significantly inhibited the increase in the DAI score and decrease in colon length in DSS-induced colitis mice.Colon histopathology revealed that the salidroside W/O/W double emulsion could alleviate the colon tissue damage,restore the structure of the colon crypt and improve the integrity of the mucosa epithelium,and reduce the infiltration of inflammatory cells,these effects are better than free salidroside.KEGG enrichment analysis of differential genes revealed that inflammation-related pathways（TNF,NF-κB,chemokine signaling pathways,etc.）,gut barrier-related pathways（tight junction and adhesion junction pathways,etc.）,and the gut microbiota were involved in the regulatory effect of salidroside W/O/W double emulsion on DSS-induced colitis in mice.The transcriptomic results were verified by detecting the expression of MPO,i NOS,NO,TNF-α,IL-1β,IL-6,IL-10,and the gut microbiota of mice.Salidroside W/O/W double emulsion contributed to the establishment of a healthy intestinal environment characterized by enhanced probiotic abundance in DSS-induced colitis mice.The main upregulated probiotics were Bacteroidetes,Actinomycetes,Proteusγand the anti-inflammatory bacteria of Enterobacteriaceae.The main downregulated harmful bacteria were Firmicutes,Campylobacter,Deferribacteraceae and Verrucamicroflora.（4）To further explore the biological activity of salidroside W/O/W emulsions after digestion in blood,an acute liver injury mouse model induced by CCl₄ was established.The differentially expressed genes were identified by transcriptomic sequencing,and the corresponding indicators were selected for validation to reveal the potential mechanism by which salidroside W/O/W double emulsion alleviated CCl₄-induced acute liver injury in mice.The results showed that salidroside W/O/W double emulsion significantly alleviated liver injury induced by CCl₄ in mice and inhibited the expression of the liver injury markers AST,ALT and GSH-ST in the serum.Compared with the CCl₄ group,the salidroside W/O/W double emulsion group exhibited 498 gene expression changes,of which 285 genes were downregulated and213 genes were upregulated,indicating that the salidroside W/O/W double emulsion could interfere with CCl₄-induced gene expression in mice liver tissues.The alleviating effect of salidroside W/O/W double emulsion on acute liver injury in mice was mainly related to the regulation of inflammatory mediators on TRP channels,cholesterol metabolism,amino acid metabolism,CYP450 enzyme metabolism on exogenous drugs,and PPAR signaling pathway.Compared with the CCl₄ group,the salidroside W/O/W double emulsion group upregulated relative expression of genes in the CYP450 family.The results indicated that CYP50 enzyme was involved in the alleviation of acute liver injury induced by CCl₄ in mice by salidroside W/O/W double emulsion.The transcriptomic results were verified by the determination of oxidative stress markers（MDA,MPO,i NOS,and NO）,antioxidant markers（GSH and GSH-PX）,and inflammatory factor expression（TNF-α,IL-1β,IL-6,and IL-10）.In summary,the construction,characterization and delivery effect of salidroside W/O/W double emulsions were studied based on the good properties of W/O/W double emulsions.Through the construction and characterization of the emulsion,the effective encapsulation and protection of the double emulsion on salidroside were confirmed.Through DSS-induced colitis in mice and CCl₄-induced acute liver injury in mice,it was confirmed that salidroside loaded in a W/O/W double emulsion could improve the relieving effect of salidroside on colitis and acute liver injury,and the delivery effect was confirmed.This study provides a basis for the further development of salidroside-related health food by extending salidroside delivery systems.