Studies On Structural Characterization And Bioactivities Of Polysaccharides From Fruits Of Lycium Barbarum L.

Objective: Aims of this project were to study the chemical structure and biological activity of polysaccharides from Lycium barbarum.As the main active ingredient in Lycium barbarum L.,the specific structure and biological activity of polysaccharide is still unknown.Therefore,in-depth study of its structure and activity may lay the foundation for the research on innovative drugs based on Lycium barbarum polysaccharides.Methods: Crude polysaccharides(LBP1)from mature fruits of Lycium barbarum were obtained using enzyme(cellulase,papain,amylase)-water extraction method.Lycium barbarum crude polysaccharide(LBP1)was separated and purified by anion exchange method(DEAE)and gel filtration chromatography(Sephacryl S-200 HR and Sephacryl S-300 HR)to achieve homogeneous polysaccharides.The molecular weight of polysaccharides was determined by high-performance gel permeation chromatography(HPGPC).The physical and chemical properties of polysaccharides and their structures were characterized by monosaccharide composition(PMP-HPLC pre-column derivatization and GC),infrared,methylation,partial acid hydrolysis,nuclear magnetic resonance spectroscopy and other physical and chemical methods.Sulfated polysaccharide Sul-LBP1B-S-2 was obtained by sulfating LBP1B-S-2 using chlorosulfonic acid-pyridine method.The tube formation of human microvascular endothelial cells(HMEC-1 cells)was employed to test anti-angiogenic activity of sulfated polysaccharide Sul-LBP1B-S-2.The toxicity effects of LBP1B-S-2 and its sulfated polysaccharide Sul-LBP1B-S-2 on HMEC-1 cells were determined by MTT assay.The content of A?42 was measured by ELISA to examine the effects of LBP1A1-1 and LBP1C-2 on the level of A?42 in the cells.The effect of LBP1A1-1 and LBP1C-2 treatment on the cell viability of Chinese hamster ovary(CHO)cells expressing APP and BACE1(CHO/APPBACE1)and Human embryonic kidney(HEK293)cells expressing APP Swedish mutantK595N/M596L(HEK293-APPsw)was tested by MTT assay and CCK-8 assay.Thioflavin T method was used to determine the effect of LBP1A1-1 on the aggregation of A?42.Tricine-SDS-PAGE employed used to examine the effect of LBP1A1-1 on A?42 monomers,oligomers and fibrils.The effect of LBP1C-2 on APP,BACE1,presenilin 1,s APP?,ADAM10,IDE and ?-actin was examined using western blotting analysis.The gene m RNA expression level was detected by RT-PCR(reverse transcription-PCR).Results:(1)Crude polysaccharide LBP1(129.0 g,yield: 2.58%)was obtained from 5.0 kg fruits of Lycium barbarum L.by enzymatic-water extraction.(2)LBP1 was isolated and purified by DEAE SepharoseTM Fast Flow column,Sephacryl S-200 HR and Sephacryl S-300 HR columns,then four homogeneous polysaccharides were obtained,designated LBP1A1-1,LBP1B-S-2,LBP1C-2,and LBP1C-5.High-performance gel permeation chromatography(HPGPC)analysis showed that the molecular weights of the homogeneous polysaccharides LBP1A1-1,LBP1B-S-2,LBP1C-2 and LBP1C-5 were 45.0 k Da,80.0 k Da,99.8 k Da and 9.8 k Da,respectively.The sugar composition of three homogeneous polysaccharides was analyzed,among them,LBP1A1-1 is a neutral polysaccharide,while LBP1B-S-2,LBP1C-2 are acidic polysaccharides.The sugar composition of four homogeneous polysaccharides was analyzed.Among them,LBP1A1-1 consisted of Rhamnose(Rha),Arabinose(Ara),Glucose(Glc)and Galactose(Gal),the molar ratio was 1.2: 47.8: 1.4: 49.8.Structural analysis showed that LBP1A1-1 is mainly composed of 1,4-?-Glc,1,3-?-Gal and 1,6-?-Gal.Its branches mainly include Terminal(T)-?-Rha,T-?-Gal,T-?-Ara,T-?-Ara and 1,5-?-Ara.The branches are linked to the C-6 position of the main chain sugar 1,3-?-Gal residue and the C-3 position of 1,6-?-Gal.LBP1B-S-2 consists of Rha,Glc A(Glucuronic acid),Gal and Ara with a molar ratio of 3.13: 3.95: 39.37: 53.55.Structural analysis showed that LBP1B-S-2 is mainly composed of 1,3-?-Gal,1,6-?-Gal and its branches mainly include 1,4-?-Glc A,T-?-Rha,T-?-Gal,T-?-Ara,T-?-Ara,1,5-?-Ara and part of 1,6-?-Gal.The branches are linked to the C-6 position of the main chain sugar residue 1,3-?-Gal and the C-3 position of 1,6-?-Gal.LBP1C-2 consists of Ara,Gal,Rha,Gal A and has a molar ratio of 57.0: 26.5: 8.2: 8.3.Structural analysis showed that LBP1C-2 is mainly composed of 1,2-?-Rha and 1,4-?-Gal A as the main chains,and the branches include T-?-Ara,1,5-?-Ara,T-?-Rha,T-?-Gal,1,3-?-Gal,1,6-?-Gal and 1,3,6-?-Gal,which are attached at the C-4 position of the 1,2,4-?-Rha backbone sugar residue.(1)CHO/APPBACE1 cells and APP Swedish mutantK595N/M596L(HEK293-APPsw)cells were treated by two kinds of homogeneous polysaccharides,LBP1A1-1 and LBP1C-2,isolated and purified from Ningxia Lycium barbarum.Biological activity results show that LBP1A1-1 and LBP1C-2 might be significantly inhibit the production of A?42 in CHO/APPBACE1 and HEK293-APPsw cells in a good dose dependent way.The results of MTT assay showed that LBP1A1-1 and LBP1C-2 had no effect on the growth of CHO/APPBACE1 cells.The results of Th T assay showed that different concentrations of LBP1A1-1 could reduce the fluorescence response of Th T to different extents,indicating that LBP1A1-1 could inhibit the aggregation of A?42.Tricine-SDS-PAGE gel was used to detect the changes of A?42 aggregation state in each group.The result showed that a large number of large molecular size fibrillar A?42 and A?42 oligomers with different degrees of polymerization and a certain amount of A?42 monomer,the content of neurotoxic neurofilament A?42 and A?42 tetramers decreased,while the amount of A?42 monomer was significantly increased.Western blotting experiments revealed that LBP1C-2 inhibited A?42 production through modulating the protein levels of the secretases involved in APP processing,or rather,inhibiting the amyloidogenic processing and promoting the nonamyloidogenic processing.Further experiments suggested that LBP1C-2 increased the expression of IDE,indicating that polysaccharides could promote A? degradation.(2)Sul-LBP1B-S-2,a sulfated derivative of LBP1B-S-2,was sulfated and its molecular weight was 131.7 k Da.Barium chloride-gelatin method measured the degree of substitution(DS)and the DS of sulfated polysaccharide was 1.42.Structural analysis showed that the sulfated substitution sites are predominantly at C-3 and C-5 of T-?-Araf and non-selectively substituted at C-2 or C-3 of 1,5-?-Araf.Biological activity results showed that the tube formation of unsulfated polysaccharide LBP1B-S-2 on HMEC-1 cells had no effect.The sulfated polysaccha- ride Sul-LBP1B-S-2 could inhibit tube formation of HMEC-1 cells even at a low concentration(0.095 ?M).Importantly sulfated polysaccharide Sul-LBP1B-S-2 showed no significant cytotoxicity effect on HMEC-1 cell.Conclusion:(1)Four homogeneous polysaccharides,namely LBP1A1-1,LBP1B-S-2,LBP1C-2 and LBP1C-5 were obtained from Lycium barbarum,respectively.(2)Among them,LBP1A1-1 is a neutral arabinogalactan.LBP1B-S-2 is an acidic arabinogalactan.LBP1C-2 is an RG-I type pectin polysaccharide.(3)Biological activity test showed that LBP1A1-1 and LBP1C-2 could not only significantly inhibited A?42 aggregation,but also significantly reduced the production of A?42.The sulfated polysaccharide Sul-LBP1B-S-2 significantly inhibited the tube formation of HMEC-1 cell.In conclusion,LBP1A1-1 and LBP1C-2 could be leading compounds that inhibited the aggregation and production of A?42,and the sulfated derivative Sul-LBP1B-S-2 could also be a leading compound that impeded angiogenesis.This project lays a solid foundation and example for the study of Lycium barbarum polysaccharides against Alzheimer's disease and angiogenesis inhibitors.