Studies On Purification, Structural Elucidation And Bioactivities Of Polysaccharides From Chrysanthemum Morifolium Flowers

The Chrysanthemum morifolium flower, a traditional Chinese medicinal herb,has been used in folk as a tea or a drug for thousands of years, and it belongs to Compositae family. In traditional Chinese medicine, it is regarded to have the effect of clearing heat and detoxification, and thus generally used to treat diseases, such as headache, influenza, hepatic and eye diseases.So far, many studies have been reported as for the chemical composition and pharmacology of Chrysanthemum morifolium flowers. However, most of them are concentrated on low molecular compounds, such as flavonoids, flavonoid glycosides,terpenes(volatile oil) and microelements. Researches on their polysaccharides are very limited, particular with respects to structural characterization and bioactivities.In this dissertation, we isolated different homogeneous polysaccharides from Chrysanthemum morifolium flowers, further elucidated their structural features and prepared their derivatives by chemical modification, such as sulfation and carboxymethylation. Finally, their biological activities were examined by using immunological model, antioxidant model and anti-angiogenesis model.In this study, the dried flowers of Chrysanthemum morifolium(3.8 kg, grown in Henan, China) were extracted successively with boiling water and 5% sodium hydroxide, followed by ethanol precipitation, respectively, yielding crude polysaccharides CMJA(295 g, yield 7.8%) and JHB(96 g, yield 2.5%). The crude polysaccharide CMJA was fractionated by anion-exchange chromatography on a DEAE-cellulose column and purified by gel permeation chromatography on a Sephacryl S-300 HR column, yielding CMJA0S1(110 mg, yield 0.4%), CMJA0S2(616 mg, yield 2.2%), CMJA1 a S2(650 mg, yield 2.3%), CMJA1 a S3(104 mg, yield0.4%), CMJA1 b S2(150 mg, yield 0.5%), CMJA1 b S3(550 mg, yield 2.0%),CMJA2S2(570 mg, yield 2.0%) and CMJA2S3(610 mg, yield 2.2%). The crude polysaccharide JHB was isolated by anion-exchange chromatography on a DEAE-cellulose column and gel permeation chromatography on a Sephacryl S-200 HR column, yielding JHB0S2(532 mg, yield 1.9%).Their structures were characterized by using monosaccharide composition analysis, linkage analysis, partial acid hydrolysis and IR, NMR spectra and so forth.CMJA0S1 was an arabinogalactan with the molecular weight of 4.7 × 104 Da. The backbone of CMJA0S2 was composed by 1, 4-β-Glcp, 1, 4-β-Galp, 1, 4-β-Manp,with the side chains of 1, 6-β-Galp, T-α-Glcp, T-α-Araf, 1, 5-α-Araf, and the molecular weight was 6.5 × 103 Da. CMJA1 a S2, CMJA1 a S3, CMJA1 b S2,CMJA1 b S3 and CMJA2S2 all belonged to pectic polysaccharides of RG-I type with different molecular weight and content of galacturonic acid. The backbone was composed of 1, 2-α-Rhap and 1, 4-α-Gal Ap, and substituted at O-4 of 1, 2-α-Rhap by arabinose and galactose residues. CMJA2S3 was the pectic polysaccharide of HG type with the molecular weight of 1.1 × 104 Da, and it was composed by 1, 4-α-Gal Ap.JHB0S2 was a xyloglucan with the molecular weight of 1.6 × 104 Da, and the backbone was 1, 4-β-Glcp with branched at O-6.The NF-κB activation test showed that these polysaccharides could activate the factor NF-κB, and probably played a key role in immunoregulatory function. By DPPH radicals scavenging assay and the protective effect on H2O2-induced PC12 cells damage, these polysaccharides were shown to exhibit antioxidant activity. In particular, CMJA0S2 exhibited the strongest antioxidant activity, with the DPPH scavenging rate and PC12 cells viability reaching 81% and 98% at the concentration of 1.0 mg/ml, respectively. In addition, JHB0S2 and its sulfated derivative JHB0S2-sul by chlorosulfonic acid-pyridine method could significantly impair the tube formation in a HMEC-1 cell model.These results demonstrate that polysaccharides from Chrysanthemum morifolium flowers possess the significant bioactivities in immune regulation, antioxidation, and anti-angiogenesis. This study can provide some insight for our understanding of the biological functions and potential use of the polysaccharides from Chrysanthemum morifolium flowers.