| Jujube contains numerous nutrients and bioactive ingredients,but the by-products produced in the processing process have not been effectively utilized.Meanwhile,there are still issues with low bioavailability and unclear functional mechanisms in the study of active substances in by-products of jujube,therefore systematic research is needed.In this work,jujube peels were used as raw material to extract jujube peel pigments(JPP)by ultrasound-assisted aqueous two-phase extraction.The extraction mechanism was analyzed using spectral measurement,microscopic observation and model fitting.The JPP was stabilized by the self-assembly of zein and the binding mechanism of JPP and zein was investigated by using multispectral and molecular simulation.Furthermore,the Caco-2 cell model and Caenorhabditis elegans(C.elegans)model were combined to evaluate the protective effect of JPP and its complex on the intestinal barrier.The main research contents and conclusions are as follows:(1)The ultrasound-assisted aqueous two-phase extraction was taken to extract JPP from jujube peels,optimizing the extraction process,and studying the extraction mechanism using spectral measurement,microscopic observation and model fitting.Under the optimal conditions of 35%(w/w)of potassium hydrogen phosphate,20%(w/w)of ethanol,1:30 g/m L of material to liquid ratio,200 W of ultrasonic power,and 50 min of extraction time,the recovery of total flavonoids in the top phase is 97.03%±1.45%,and the distribution coefficient is 5.97±0.21.Rutin,quercetin3-β-D-glucoside and kaempferol-3-O-rutinosid were identified as the main composition of JPP by UPLC-MS/MS.Results of the extraction mechanism study showed that the salting-out effect,hydrogen bond force and van der Waals force promoted the mass transfer of solvent to jujube peel cells,and then the extraction behavior occurred.After extraction,the average particle size of the top phase significantly increased from 83.13±0.70 nm to 116.87±1.69 nm.The mechanical destruction of ultrasound on jujube peel cells further accelerated the release of flavonoids,which were captured by W/W emulsion in the bottom phase and distributed to the top phase.Compared with ethanol extraction,JPP from ultrasound-assisted aqueous two-phase extraction contained a higher extraction yield.The extraction process is exothermic and spontaneous,which conforms to the first-order kinetic model.(2)Zein was used to stabilize JPP,and the binding mechanism during the stabilization process was elucidated through multi-spectra and molecular simulation techniques.The JPP/zein complex(JZC)was prepared by the anti-solvent precipitation method,and the optimum material ratio was selected through the loading efficiency and particle size potential.When the concentration of JPP is 60μM,the loading efficiency reached 68.30%±1.50%.The particle size of the nanoparticles is uniform,and the system is stable.The complex contains a slow-release effect,in which the release curve conforms to the Korsmeyer-Peppas model.The bioavailability is significantly enhanced to39.56%±2.56%.The interaction types of the JZC was studied using multiple spectral techniques.It was found that the formation of the complex was a static quenching process with high affinity,and the amino acid microenvironment of zein were changed.The results of circular dichroism analysis indicate that after binding with JPP,theα-helix content of zein increased from 56.40%to59.40%,with a tight binding.Computer simulation technology was used to further explore the bond mechanism of nanocomposites.Molecular docking provides the optimal prediction model of nanocomposites.Through molecular dynamics simulation,tryptophan residue Q215 in zein provides the maximum binding free energy(-2.42±0.32 kcal/mol)during the formation process of JZC.In summary,after embedding with zein,the stability and bioavailability of JPP are significantly improved,and the two are tightly bound through hydrophobic interactions.(3)The Caco-2 cell monolayer model and C.elegans model were constructed to study the protective effect and pathway mechanism of JPP and its complex on lipopolysaccharide(LPS)induced intestinal barrier damage.Caco-2 cell monolayer was used to simulate the intestinal barrier.JPP and its complex contain protective effects on LPS-induced intestinal barrier damage.After incubation with JPP/zein complex,the membrane potential of Caco-2 cell monolayer was enhanced to 500.61±30.94Ω·cm~2 significantly,improving the permeability.After incubation with JPP/zein complex,the relative expression levels of Claudin-1,Occludin,and ZO-1 in Caco-2 cells were maintained at 85.55%±5.47%,66.52%±6.70%,and 87.96%±3.36%,respectively,activating tight junction proteins and alleviating intestinal barrier damage.JPP and its complex are non-toxic to C.elegans and have no intestinal damage.The intestinal integrity of C.elegans could be improved by the incubation of JPP and its complex.Meanwhile,the complex can significantly reduce the accumulation of lipofuscin caused by LPS in C.elegans.The number of DEPs in C.elegans increased significantly after the culture of JPP and its complex.Furthermore,the lysosome pathway of C.elegans was activated to further regulate immune response and lipid transport,improving the intestinal barrier function of C.elegans.The results of combining the Caco-2 cell monolayer model and the C.elegans model showed that both JPP and its complexes have a protective effect on intestinal barrier damage,with the JPP/zein complex having a more significant effect.In summary,the establishment of ultrasound-assisted aqueous two-phase extraction for the efficient preparation of JPP resulted in a 75.25%increase in total flavonoid yield compared to conventional ethanol extraction.The stable treatment of JPP using zein through the anti-solvent precipitation method increased the bioavailability of JPP by 34.28%.The JZC exerts its intestinal barrier protective effect by regulating the tight junction protein in Caco-2 cells and the lysosome pathway in C.elegans. |
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