Study On Extraction And Biological Activity Of Polysaccharides From Glycyrrhiza Residue

Glycyrrhiza is one of the commonly used Chinese herbal medicines.The average annual consumption of glycyrrhiza in our country exceeds 60,000 tons.It is mainly used for processing into glycyrrhizic acid and glycyrrhiza extract,and a large amount of glycyrrhiza residue is produced during the processing.A small part of glycyrrhiza residue is used as a cultivation substrate,and most of them are landfilled,incinerated or piled up in the open air,resulting in waste of glycyrrhiza resources and environmental pollution.It has been reported in the literature that glycyrrhiza residue contains 2.0%-2.5%polysaccharides,which have antioxidant,hypoglycemic,and immune regulation effects.In this thesis,using glycyrrhiza residue as raw material,the enzyme-assisted two-phase salting-out extraction technology was used to extract glycyrrhiza residue polysaccharides,and the polysaccharides of glycyrrhiza residue were separated and purified to explore their physicochemical properties and biological activities.The specific research contents are as follows:First,the extraction process of polysaccharide from glycyrrhiza residues was studied by enzyme-assisted two-phase salting-out extraction.The effects of enzyme type,enzyme amount,enzymolysis pH,enzymolysis temperature and enzymolysis time on the yield of polysaccharide were investigated,and the optimal enzymolysis conditions were obtained through response surface optimization.The results showed that under the conditions of cellulase andβ-glucanase ratio of 2:1,enzyme amount of 820 U/g,enzymolysis pH 4.0,enzymolysis temperature 50℃and enzymolysis time 120 min,the yields of polysaccharides in the top and bottom phases were the highest,which were 0.88±0.04%and 2.58±0.08%,respectively.The yields of polysaccharides from glycyrrhiza residue were compared between the microwave/enzyme/microwave-enzyme coupled salting-out extraction technology and the hot water method.Secondly,the physicochemical properties,structure and biological activity of the top-phase polysaccharide（GRP-1）and the bottom-phase polysaccharide（GRP-2）extracted by enzyme-assisted two-phase salting-out extraction were studied.The results show that GRP-1 and GRP-2 are significantly different in main chemical composition,monosaccharide composition,triple helical structure,microscopic morphology,crystal structure and thermal stability;GRP-1 exhibits strong·O₂^-scavenging,while GRP-2has strong DPPH,ABTS,·OH scavenging ability and restorative ability;GRP-2 has strong inhibitory ability onα-amylase andα-glucosidase;GRP-1 and GRP-2 has no toxicity to macrophage RAW 264.7,can induce the release of NO and cytokines（IL-1β,TNF-αand IL-6）,showing strong immunomodulatory effect.Thirdly,GRP-1 and GRP-2 were separated and purified,and the physicochemical properties and biological activities of each component of the purified top and bottom polysaccharides were studied.The results showed that GRP-1 and GRP-2 were purified by DEAE-52 cellulose column to obtain three components respectively;The monosaccharide composition shows that GRP-1-a is a heteropolysaccharide mainly composed of glucose,GRP-1-b is mainly composed of glucose,fucose,galacturonic acid and galactose,and the content of galacturonic acid and glucose in GRP-1-c is 90.58%;GRP-2-a and GRP-2-b are mainly composed of glucose,galacturonic acid,xylose and fucose,and the content of galacturonic acid and xylose in GRP-2-c is 91.05%;The inhibitory ability of the top phase and bottom phase polysaccharide components toα-amylase was significantly enhanced,and had no toxicity to macrophage RAW264.7,could enhanced phagocytic activity,and promoted the release of NO and cytokines（IL-1β,TNF-αand IL-6）.Finally,the digestibility of three top phase polysaccharides components after digestion with simulated saliva-gastric-intestinal,and the changes of their monosaccharide composition,characteristic groups and microscopic morphology were studied.The results showed that the reducing sugar content of the three polysaccharides increased significantly after digestion,indicating that the polysaccharides were partially degraded,among which the lowest digestibility of GRP-1-b was 7.30%;The anti-glycation activities of the three bottom phase polysaccharides components were investigated.The results showed that the highest inhibition rate of GRP-2-b on fructosamine was 57.2%,and GRP-2-a had the strongest inhibitory effect onα-dicarbonyl compounds.GRP-2-a and GRP-2-b could effectively inhibit the formation of advanced glycation end products（AGEs）with a good anti-glycation effect.