| Glycyrrhiza uralensis Fisch.is an important Chinese herbal medicine in Gansu Province.A large number of studies have confirmed that Glycyrrhiza uralensis polysaccharide has immunomodulatory,antiviral and antitumor effects,which is of great research significance.To further study the bioactivity of the liquorice polysaccharide,a more efficient extraction method was designed,this paper used ultrasound-assisted extraction of Glycyrrhiza uralensis polysaccharides（GP）and established a polysaccharide extraction kinetic model to analyzed the structural characteristics and physical and chemical properties of GP;GP has been proven to have good antiviral properties,so its activity against Bovine Viral Diarrhea Virus（BVDV）was studied on this basis;the research showed that GP has a good anti-BVDV effect,and polysaccharide microencapsulation have been widely studied and applied as the current research trend,on these basis,Glycyrrhiza uralensis polysaccharides microcapsules（GPM）were prepared to studied its effect on wound healing in SD rats and confirmed the feasibility of microencapsulation of GP.The main results are as follows:（1）By analyzed the single factor experiment and response surface method to confirme the optimal conditions of ultrasonic-assisted extraction,and verified the yield of soluble polysaccharides under the optimal extraction conditions（80℃,140 minutes,1:11）is 22.31±0.07%.According to Fick’s second law,the GP extraction process model equation was established,and the thermodynamic analysis showed that the ultrasonic-assisted extraction process of GP was an irreversible endothermic process.HPLC-DAD-ELSD,Fourier transform infrared spectroscopy（FT-IR）,circular dichroism（CD）measurement,thermo gravimetric analysis/differential thermal analysis（TGA/DTA）were used to determined the physical and chemical properties of GP,the results showed that GP is a non-reducing glycoprotein polymer with phosphonic acid and sulfate ion groups,a helical sheet structure,and high thermal stability.According the study of the influence of GP solution mass concentration,pH,different ion species and concentration,and the temperature on the apparent viscosity of GP solution found that the absorbance of GP increased significantly with temperature,and had a maximum at pH 5.5,then decreased with the pH from 6.0 to 13.0;the apparent viscosity of GP increased with the increase of the mass concentration of the GP solution;when the temperature exceeded at 60℃,the viscosity of the GP solution dropped sharply,the minimum viscosity was 0.14Pa·s,and with the stirring rate increased,the apparent viscosity of the GP solution decreased significantly;the apparent viscosity of the GP solution increased with the increase of Na+and K+concentrations,and decreased significantly with the increase of Ca2+and Zn2+,and showed a concentration dependence.Scanning electron microscope（SEM）and atomic force microscope（AFM）were used to observed the characteristic structure and morphological characteristics of GP.It can be seen that the GP molecule was irregular spherical granular with a helical structure.FT-IR analysis shows that GP isα-D-pyran sugar.（2）The virus gradient and virus growth value detection,CCK-8 cytotoxicity test,qRT-PCR were used to determined the relevant mRNA expression in BVDV and MDBK cells,IRF-1 and IRF-3 were used to regulated the activity in MDBK cells as the influence on BVDV indicators to studied the antiviral properties of GP.The results showed that both RBV and GP had good anti-BVDV activity;GP could significantly inhibited the gene expression of IRF-1（##p<0.01）,and the gene expression fold was reduced to a minimum when the GP concentration was 200μg/mL;both GP and RBV could promoted the expression of IRF-3,through up-regulated the production of IFN-αby IRF-3 signal-mediated,it could produced antiviral activity against BVDV.It was concluded that GP had a better preventive effect on the BVDV virus.（3）According to the optimized process,the optimal GPM combination conditions were selected as sodium alginate concentration 2%,chitosan concentration 0.3%,GP concentration0.6g/mL,calcium chloride concentration 3%,which had the maximum drug loading rate and embedding rate were 42.8±0.74%and 59.92±0.68%,respectively.GPM had a good swelling properties,the swelling rate increased linearly with time from 10 to 30 minutes,and the swelling equilibrium was reached in 36 minutes.It could be seen from TEM and SEM images that GPM exhibited good dispersion characteristics and relatively dense structure,with a flat surface and good separation from the mixture.It could effectively wrapped the soluble polysaccharide molecules in the carrier to avoid drug leakage and ensured the drug carrier system storage;its three-dimensional network structure was conducive to cell adhesion,proliferation and tissue growth;layered GPM with a small amount of short chain branches had a loose and soft fiber structure and a dry surface.The characteristic peaks of GPM were studied by FT-IR,and the results showed that GPM contains multiple hydroxyl（-OH）groups,C-H,C=O and C-O-C,O-Si.The XRD curve showed that GPM has three sharper peaks at15.20°,20.16°,and 22.25°than GP.It can be considered that GPM was affected by physical grinding and the chemical substances added in the preparation of microcapsules,changing from a relatively ordered crystal structure to disordered structure.The weight loss and thermodynamic processes of GPM were analyzed by TG/DSC spectroscopy,which showed that GPM may have loose texture,rough surface,and poor thermal stability.Through the measurement of physical properties,it could be known that GPM shows small hardness（2.0±0.157）,medium viscosity（0.0372±0.00599）and elasticity（0.18±0.0258）,and can be used as an ideal biomedical material.（4）Through the detection and analysis of blood routine indexes and blood biochemical indexes,it was found that the hemoglobin（HGB）,red blood cell volume（HCT）and mean red blood cell hemoglobin concentration（MCHC）of the GPM group were significantly higher than those of the control group（p<0.05）,proving that GP could reducedwound inflammation.Observation of the granulation tissue changes in the SD rat injury model found that the granulation tissue of the GPM group showed obvious proliferation of blood vessels and fibroblasts,with the thickest arrangement of collagen fibers,and its microvessel count was slightly higher than the ferulic acid group,which was presumed to be GP played an important role in promoting the formation of capillaries.Compared with other groups,the hydroxyproline content in the GPM group increased significantly（p<0.05）,and the relative expression of VEGF and mi RNA-21 genes increased significantly（p<0.01）,indicating that GP could regulated this gene.In the western blotting results,the expression of p-STAT3 in the granulation tissue of the GPM group was significantly higher than the others,indicating that GP could activated the protein expression of STAT3 and further regulated the gene expression of VEGF and miRNA-21,thereby inducing the form of neovascularization.The above research results show that GPM has a significant effect on healing damaged tissues in rats. |
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