Isolation,Purification,Structural Features And Anti-Inflammatory Activity Of Polysaccharides From Blackened Jujube

Jujube(Ziziphus jujuba Mill.),as a medicinal food product,has high nutritional and medicinal value.Jujube polysaccharide is an important active component for its efficacy and have various biological activities such as anti-inflammatory,anti-oxidant,anti-cancer and liver protection.Blackened jujube is a product obtained by solid-state fermentation of red jujube fruit,which has higher nutritional value and processing prospect.Studies related to the treatment of colitis with jujube polysaccharides have been reported,but little research has been reported about the protective effects and mechanism of blackened jujube polysaccharides on colitis.In the present study,a blackened jujube polysaccharide(BJP-4)was obtained using isolation and purification techniques,its structure was characterized,and the anti-inflammatory activity of BJP-4 was analyzed by constructing a cell inflammation model and a mouse colitis model.The specific study results are as follows:(1)Isolation and purification of blackened jujube polysaccharideThe crude polysaccharide was produced by ultrasonic-assisted hot water extraction,and then a purified fraction was obtained by degreasing,protein removal,alcohol precipitation,DEAE-52 cellulose column,Sephadex G-100 dextran gel column and freeze-drying,and named as BJP-4.The total sugar content of BJP-4 was 95%±0.72%,and the ultraviolet(UV)spectrum showed that it contained very little nucleic acids and proteins.(2)Structural characterization of BJP-4The molecular weight(Mw)and monosaccharide composition of BJP-4 were determined by high performance gel permeation chromatography(HPGPC)and high performance anion exchange chromatography(HPAEC),respectively,which showed that BJP-4 was mainly composed of galacturonic acid,arabinose,galactose and glucose with a mass ratio of49.40:33.54:7.71:7.19 and an Mw of 1.24×10~5Da.The combined results of fourier transform infrared spectrum(FT-IR),nuclear magnetic resonance(NMR)and methylation showed that BJP-4 was dominated by→4)-α-L-Galp A(1→,→5)-α-L-Araf(1→and→4)-β-D-Galp(1→with bothα-andβ-type glycosidic bonds.The results of advanced structural analysis showed that BJP-4 has a spherical mass shape under atomic force electron microscopy(AFM),showing molecular aggregation effects,and both crystalline and non-crystalline structures under X-ray diffraction(XRD),indicating no triple helix structure under circular dichroism(CD).(3)In vitro anti-inflammatory activity of BJP-4The anti-inflammatory capacity of BJP-4 was analyzed by establishing a lipopolysaccharide(LPS)-induced inflammation model in RAW264.7 cells.The results indicated that BJP-4 dose-dependently alleviated cell inflammation through inhibiting Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB)pathway,regulating the release of inflammatory cytokines and modulating the expression of related enzymes.This was evidenced by inhibition of NO secretion,reduction of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and IL-1βlevels and m RNA expression,as well as reduction of inducible nitric oxide synthase(i NOS)and cyclooxygenase-2(COX-2)related m RNA and protein expression levels.(4)The anti-inflammatory activity of BJP-4 in vivoBy establishing a dextran sodium sulfate(DSS)-induced ulcerative colitis(UC)mice model,BJP-4 was found to have a certain in vivo anti-inflammatory activity.BJP-4 alleviated DSS-induced colitic symptoms through altering gut microbiota structure,modulating TLR4/Myeloid differentiation factor 88(My D88)/NF-κB/NOD-like receptor thermal protein domain associated protein 3(NLRP3)pathway,rebalancing pro/anti-inflammatory cytokines,improving oxidative status,enhancing the integrity of the intestinal epithelial barrier and promoting the production of Short chain fatty acids(SCFAs).During the experiment,it was observed that BJP-4 intervention significantly slowed weight loss,increased thymic index,decreased splenic index,reduced DAI scores,and inhibited colonic shortening compared to the model group.H&E staining results showed that BJP-4 could repair epithelial barrier damage and prevent inflammatory cell infiltration.BJP-4 could also regulate oxidative status,increase superoxide dismutase(SOD)and catalase(CAT)activities,and decrease malondialdehyde(MDA)production.BJP-4 was found to increase the m RNA expression levels of band closure protein-1(ZO-1)and mucin-2(Muc-2)by RT-PCR techniques.Western Blot analysis showed that BJP-4 significantly upregulated the levels of TLR4,My D88,NLRP3 and Apoptosis-associated speck-like protein(ASC)as well as decreased the p-NF-κB/NF-κB ratio.The determination by gas chromatography-mass spectrometry(GC-MS)revealed that BJP-4 could increase the content of SCFAs,such as acetic acid,propionic acid and butyric acid.The 16S r RNA gene sequencing results suggested that BJP-4 was able to increase theαandβdiversity of the intestinal flora of mice with colitis,while modulating the composition of intestinal microorganisms at the phylum and genus levels,increasing the number of beneficial bacteria and decreasing the abundance of harmful bacteria.