Studies On Isolation, Purification, Structural Elucidation And Biological Activities Of Polysaccharides From Glycyrrhiza Inflata Bat. In Xinjiang

As an important traditional Chinese medicine, the roots and rhizomes of Glycyrrhiza spp. have been used for centuries with the activities of heat-clearing and detoxifying, invigorating the spleen and replenishing qi, moistenning lung, relieving cough and alleviate the drug property, etc. Glycyrrhiza spp. are also the ethnic medicine resource with the reginal characteristics and the famous medicinal materials in Xinjiang. Morden researches of chemistry and pharmacology of Glycyrrhiza spp. have focused on the chemical constituents, such as triterpenoids and flavonoids. Meanwhile, there are less researches of another active constituent-Glygyrrhiza polysaccharide(GPS). We have studied for the first time the structural elucidation, biological activities and structure-activity relationships of polysaccharides from Glycyrrhiza. inflata Bat.-the main resource of Xinjiang licorice.ObjectiveTo systematically study the polysaccharides from Glycyrrhiza inflata Bat. in Xinjiangand clarify the chemical structure, biological activities and structure-activity relationship of the polysaccharides, which will provide scientific basis for new polysaccharide drug research and comprehensive utilization of Glycyrrhiza polysaccharide.Methods1 . Extraction, isolation and purification of GPS. Water extracting-alcohol precipitating method was applied to extract crude polysaccharide and dialysis and DEAE cellulose ion-exchange chromatography were applied to fractionalize. Every fraction was purified by Sepharose and Sephadex gel chromatography.Purity and molecular weight of every homogeneous polysaccharide were determined by HPGPC method.2 . Structural elucidation of homogeneous polysaccharide.Based on chemical methods (including sugar compositin analysis, methylation analysis, uronic acid reduction, partial acid hydrolysis and enzymatic digestion) and spectral analysis (IR, NMR,GC-MS), the structural characteristics of homogeneous polysaccharide was investigated.3.Study on the biological activities of GPS.Mouse splenocyte proliferation assay in vitro, free-radicals scavenging assay and antiblastic assay of cancer cell in vitro.were applied respectively to investigate the immunological activity, antioxidation activity and antitumor activity.4.Study on the activity-structure relationship of active polysaccharides. Partial acid hydrolysis, uronic acid reduction and enzymatic digestion were used to treat the active polysaccharides from Glycyrrhiza Inflata Bat.To compare the immunostimulating and antitumor activities between the structural fragments and the original polysaccharides and clarify the active fragments and structure-activity relationship.Results1.The roots and rhizomes of Glycyrrhiza Inflata Bat.were extracted with distilled water and precipitated with ethnol. The deproteinized and decolored residues were separated and purified by DEAE ion-exchange chromatography and Sepharose CL-6B, Sephadex G-50 gel-filtration chromatography repeatedly. Thirteen homogeneous polysaccharides were obtained for the first time from Glycyrrhiza inflata Bat. Their properties and structures were identified on the basis of chemical methods and spectral methods. Among the 13 homogeneous polysaccharides, Gi-A1 and Gi-A2 were neutral arabinogalactoglucan; Gi-A3 and Gi-A4 were neutral arabinogalactan; Gi-B1,Gi-C1,Gi-D1 were typical RGⅠtype pectic polysaccharides,which contained a backbone of the repeating disaccharide[→4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→],with substitution at O-4 of the rhamnopyranosyl residues; the rest included 2 neutral complicated heteropolysaccharides and 4 acidic complicated heteropolysaccharides.2.The results of the biological activities indicated that the crude GiP and its purified fractions had remarkable immunostimulating activities, especially the H2O and lower concentration NaCl eluate fractions from DEAE ion-exchange chromatography and their purified fractions. The results of antitumor tests on GiP and its purified fractions showed only the higer concentration NaCl eluate fractions had certain antitumor activities. Additionally, GiP and some purified fractions showed certain activities on scavenging free-radicals, such as DPPH·,·OH, and O2·, which indicated they would have antioxidation activity.3.Compared with their reduced products, partial hydrolysis products and enzymatic digestion products, three typical pectic polysaccharides Gi-B1, Gi-C1 and Gi-D1 have showed more potent biological activities. The results of the studies on the relationships between the structures and the activities of the active polysaccharides from Glycyrrhiza inflata Bat.indicated that the neutral glycosyl side chains, the uronic acid unit and the rhamnogalacturonan core of the active pectic polysaccharide were each involved in the expression of biological activities.Conclusion1.13 homogeneous polysaccharides were obtained from Glycyrrhiza inflata Bat.and their structure were elucidated for the first time.All of them were complicated heteropolysaccharides with multi-branch,including arabinogalactoglucan, arabinogalactan and pectin possessed anα-(1→4)-galacturonan backbone with some insertions ofα-(1→2)-rhamnopyranosyl residues.2.Crude and homogeneous polysaccharides from Glycyrrhiza inflata Bat.had various biological activities.They could stimulate mouse slenocyte proliferation in vitro, inhibitate the growth of some cancer cell in vitro and scanvenge free-radicals, such as DPPH,·OH, and O2·.3.Some active fragments of pectic polysaccharides from Glycyrrhiza inflata Bat. were revealed. The neutral glycosyl side chains, the uronic acid unit and the rhamnogalacturonan core of the active pectic polysaccharide were each involved in the expression of biological activities.For the first time, the structural characterizations and activities of polysaccharides of Glycyrrhiza inflata Bat. were reported. These researches enriched the studies on chemical constituents of Glycyrrhiza inflata Bat., filled the gaps in the studies on the polysaccharide of Glycyrrhiza inflata Bat., identifed the chemical structure, biological activities and the structure-activity relationship of the polysaccharides from Glycyrrhiza inflata Bat., which would provide research data for clarifying the efficient substance and for the comprehensive utilization of Glycyrrhiza.