Extraction Optimization,Structural Characterization,and Biological Functions Of Polysaccharides From The Leaves Of Lithocarpus Litseifolius

Lithocarpus litseifolius is an evergreen tree belonging to the genus Fagaceae.Its leaves are of the same origin as food and medicine,and can be used as tea,sugar and medicine.Polysaccharide,as the mainly active component of L.litseifolius,has antioxidant,hypoglycemic,antiglycation and other health effects.In general,the biological activities of natural polysaccharides are closely related to their structural characteristics.However,the structure and potential structure-activity relationship of L.litseifolius polysaccharides are still unclear,which limit the development and application of L.litseifolius polysaccharides in the functional food industry.Therefore,in order to further promote the processing and functional product development of L.litseifolius polysaccharides,the extraction technology of L.litseifolius polysaccharide was firstly optimized in this study,namely,the pressurized hot water assisted extraction and microwave assisted deep eutectic solvent extraction.Then,the polysaccharides were isolated and purified,and the physicochemical characteristics of the purified polysaccharides were analyzed,and their potential structure-activity relationships were discussed.The main research results are as follows:（1）Firstly,the conditions of pressurized hot water extraction and microwave assisted deep eutectic solvent extraction were optimized,and the extraction yields of L.litseifolius polysaccharides was evaluated.The single factor experiment and response surface experiment were used to evaluate the effects of extraction pressure,extraction time,and liquid-solid ratio of pressurized hot water extraction on the yield of polysaccharides from L.litseifolius.Besides,the effects of water content of DES,microwave extraction power,microwave extraction time,and liquid-solid ratio of microwave assisted deep eutectic solvent extraction on the extraction yield of polysaccharides from L.litseifolius were also evaluated by the single factor experiment and response surface experiment.The results showed that the optimal extraction conditions of pressurized hot water extraction were as follows:liquid-solid ratio 22 m L/g,extraction time 40min,extraction pressure 0.19 MPa.Under these conditions,the actual extraction rate was 4.64%±0.03%.The optimum conditions of microwave assisted deep eutectic solvent extraction were as follows:water content of DES was 21.0%,extraction power was 576 W,extraction time was11 min,liquid-solid ratio was 29.0 m L/g.Under these conditions,the actual extraction rate of polysaccharides was 4.16%±0.09%.Compared with the traditional hot water extraction method（extraction yield of 3.46%±0.11%）,the two methods optimized in this study have higher extraction efficiency.The results of this study are expected to provide technical support for the processing of polysaccharides from L.litseifolius.（2）In this study,the fractional alcohol precipitation combined with membrane separation technology was used to efficiently separate polysaccharides from L.litseifolius prepared by three extraction technologies.Then,the physicochemical characteristics of the purified L.litseifolius polysaccharides were analyzed.The high performance size exclusion chromatographic analysis showed that polysaccharides prepared by three different methods all exhibited homogeneous and symmetrical peaks,and the molecular weight of polysaccharides prepared by traditional hot water extraction（P-W）,pressurized hot water extraction（P-P）and microwave assisted deep eutectic solvent extraction（P-DM）was 25.41×10⁴ Da,22.29×10⁴Da and 15.75×10⁴ Da respectively.The total sugar content of P-W,P-P and P-DM was 81.30%±1.40%,87.29%±0.72%and 85.48%±0.36%respectively,the uronic acid content was 38.19%±0.41%,44.29%±0.29%and 43.43%±0.93%respectively,the content of conjugated phenolic compounds was 20.36±0.75 mg GAE/g,13.98±0.72 mg GAE/g and 43.26±0.69 mg GAE/g respectively,.the protein content was 7.49%±0.10%,6.03%±0.28%and 4.32%±0.05%,respectively,and the esterification degree was 38.22%±1.32%,36.71%±1.06%and 31.23%±0.59%,respectively.The types of monosaccharides of L.litseifolius polysaccharides prepared by three extraction methods were the same,while their molar ratios were varied in different extraction methods.They were mainly composed of galacturonic acid,galactose,and arabinose,indicating that L.litseifolius polysaccharides were acidic polysaccharides.Furthermore,the infrared spectra and nuclear magnetic resonance spectra showed that the primary chemical structures of L.litseifolius polysaccharides preapared by different extraction methods were similar,which possessed the same types of sugar residues,such as 1,5-α-L-Araf,T-α-L-Araf,1,4-α-D-Gal Ap,1,4-α-D-Gal AMep,and 1,3,6-β-D-Galp,etc.it was inferred that L.litseifolius polysaccharides were rich in homogalacturonan and arabinogalactan.（3）Rresults showed that L.litseifolius polysaccharides prepared by different methods showed good antioxidant activities（ABTS scavenging activity,DPPH scavenging activity and NO scavenging activity,and total reducing power）,antiglycation activities,hypoglycemic activities（α-glucosidase inhibition）,immunomodulatory activities,and good in vitro prebiotic activity.In particular,compared with P-W and P-P,P-DM showed better biological activities.The IC₅₀ value of ABTS scavenging ability of P-DM was 0.857±0.004 mg/m L.The IC₅₀ value of DPPH scavenging ability of P-DM was 1.580±0.040 mg/m L.The IC₅₀ value of NO radical scavenging activity P-DM was 0.812±0.013mg/m L.The IC₅₀ value of antiglycation activity of P-DM was 0.669±0.008 mg/m L.The IC₅₀ value ofα-glucosidase inhibitory activity of P-DM was 4.629±0.009μg/m L.This may be closely related to its higher content of uronic acid,higher content of conjugated phenolics,lower degree of esterification,lower molecular weight and other structure characteristics.The findings in this study are helpful to clarify the structural characteristics and potential structure-activity relationship of L.litseifolius polysaccharides,and are expected to provide scientific basis and technical support for the processing and utilization of L.litseifolius polysaccharides.