| Umbilicaria esculenta(Chinese name “Huangshan Shier”),which grows in Huangshan Mountain in Anhui,is a type of edible and pharmaceutical lichens.Polysaccharides are the main bioactive components of U.esculenta,which can enhance the immunoregulatory,anti-tumor,anti-virus and antioxidant activity.In this paper,we investigated the extraction and purification,structural characterization and pharmacological activity of polysaccharide from U.esculenta by chemical and instrumental analysis,structural characterization methods,molecular modification methods and biological methods.The research results were as following:(1)The degreased powder of U.esculenta was extracted by hot water,deproteinization and dialysis to give the crude polysaccharide UP.The extraction yield was 5.68%.Further,the crude UP was fractionated by DEAE-Cellulose ion-exchange chromatography and purified by Sephacryl S-200 gel filtration chromatography to obtain three homogeneous polysaccharide fractions: UP1-1,UP2 and UP3.Their molecular weights were estimated to be 2.81 × 105 Da,3.33 × 105 Da and 5.05 × 103 Da,respectively.Monosaccharide composition analysis,methylation analysis and NMR results showed that UP1-1 mainly consisted of mannose,glucose and galactose in ratio of 1.0: 4.6: 0.8,which may contain traces of arabinose.The backbone of UP1-1 consisted of 1,6-?-D-Glcp,1,4-?-D-Glcp and 1,6-?-D-Manp with the branched chains 1-?-D-Glcp,1,3-?-D-Galp and 1-?-D-Galp substituted at O-3 position of 1,6-?-D-Manp.The monosaccharide composition of UP2 was mainly mannose,glucose and galactose in the molar ratio of 1.7: 1.0: 1.2,and the backbone of UP2 consisted of 1,6-?-D-Glcp and 1,6-?-D-Manp with the branched chains 1,6-?-D-Galp and 1-?-D-Galp substituted at O-3 position of 1,6-?-D-Glcp.UP3 was mainly composed of mannose,glucose and galactose in the molar ratio of 2.5: 1.9: 1.8,there may be a small amount of rhamnose,arabinose,xylose.Structure analysis showed that the backbone of UP3 consisted of 1,6-?-D-Glcp,1,3,6-?-D-Manp and 1,3,4-?-D-Xylp,branched chains were 1-?-D-Galp,1-?-L-Rhap and 1,3-?-L-Arap.(2)The chlorosulfonic acid-pyridine method and mixed phosphate reagent method were employed to modify UP2,respectively.The results of sulfation modification showed that UP2 was modified to obtain a sulfated fragment UP2-S with a maximum degree of substitution of 1.67 and its molecular weight was 2.45 × 107 Da.FT-IR of UP2-S was indicated that the characteristic peaks at 1250 cm-1,813 cm-1 and 588 cm-1 were due to the S=O glycosidic,C-O-S glycosidic and O-S-O glycosidic vibration,respectively.The results confirmed that the sulfate group successfully combined with polysaccharide UP2.The results of phosphorylated modification were shown that nine phosphorylated fragments(UP2-P)were obtained.The effects of reaction temperature,reaction time and reaction pH on the degree of substitution(DS)of polysaccharide were investigated by single factor test.On the basis of the results of single factor test,the reaction systems of phosphorylation were further optimized by orthogonal design.The maximum degree of substitution of 0.836 was obtained under an optimal reaction conditions of orthogonal design test including phosphorylating agent,pH=7,the reaction temperature of 90 ?,and the reaction time of 5 h.The results of FT-IR were demonstrated that the characteristic absorption peaks at 1260 cm-1 and 1110 cm-1 were attributed to the stretching vibration of P=O and P-O-C bond,respectively,which had confirmed the success of phosphorylation of UP2.(3)The immunoregulatory activity and antitumor activity of the polysaccharide fragments were investigated by biological activities experiments.In vitro macrophage immunomodulatory assay showed that all the polysaccharide fractions(UP1-1,UP2 and UP3)could increase the cell proliferation,neutral red phagocytic activity and the yield of NO of RAW 264.7 cells.The anti-tumor activities of polysaccharide(UP1-1,UP2 and UP3)indicated that the polysaccharide fractions had different inhibitory activities on liver cancer cells(Hep G2).At all concentrations,the UP2-S significantly inhibited antitumor activity of Hep G2 cells.At the concentration 200 ?g/m L,UP2-S had the highest inhibition rate reaching 50.36%,which was 2.4 times of the inhibition rate of UP2 at the same concentration.The anti-tumor activities experiments indicated that different substitution degree of UP2-P had different anti-tumor activity against Hep G2 cells.The UP2-P5 had the best anti-tumor activity at all concentrations,and the highest inhibitory rate was reach 40.80%.The results of the study have a certain guiding significance for the development and application of polysaccharides from U.esculenta grown in Huangshan Mountain. |
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