| Background: Polygonati Rhizoma(Polygonatum cyrtonema Hua,Polygonatum sibiricum or Polygonatum kingianum)was first recorded in “Mingyibielu” of the Han Dynasty which was believed to stimulate saliva secretion,replenish energy,relieve inflammation and enhance immunity.The "Nine-Steam-Nine-Bask" method is the best way to increase the benefit of the function of Polygonati Rhizoma.Objective: In this paper,the physicochemical properties and the qualitative and quantitative analysis of the crude polysaccharide from processed Polygonatum cyrtonema Hua.were carried out.We combined antioxidant experiments to optimize the beststeamed P.cyrtonema polysaccharides(PCPs).The anti-inflammatory activity of processed PCPs was verified by MH7 A human arthritis synovial fibroblasts.Then the most effective activity of processed PCPs was separated and purified.Subsequently,the primary structure and antioxidant activity of polysaccharides(PCP-F1)were analyzed.Methods: In this study,the "Nine-Steam-Nine-Bask" P.cyrtonema were prepared by steaming for 12 hours and basking for 12 hours for nine consecutive times,and crude processed P.cyrtonema polysaccharides were extracted using water extraction and precipitated in ethanol.The physicochemical properties of P.cyrtonema and its polysaccharides were characterized by UV,HS GC-MS,total sugar content,acid sugar content,p H value,HPLC-GPC,IR and NMR.Combined with antioxidant activity and MH7 A cell experiment,the anti-inflammatory activity of steamed P.cyrtonema polysaccharides was investigated,and the best steamed polysaccharide was selected.The crude polysaccharide was further separated and purified to obtain refined polysaccharide(PCP-F1).Its primary structure was analyzed by means of UV,GC-MS,HPLC-GPC,IR,1D&2D NMR and Congo red analysis.The antioxidant activity of PCP-F1 was determined.Results: The color and appearance of processed P.cyrtonema gradually changed from yellow to black,and then to dark black as the steaming times increased.HS GC-MS detected acetic acid and 5-methyl furfural content with steam times that reached their highest at 6-Z and then decreased gradually;5-Z was the highest level of furfural,followed by a gradual decline in contents.The total sugar content showed a cyclical downward trend in P.cyrtonema after steaming.During the steaming process,the contents of galactose and glucose were higher,and the high molecular weight polysaccharide was partially hydrolyzed,and the Maillard reaction occurred to form small polysaccharides,monosaccharides and trace oligosaccharides.NMR analysis indicated that 5-hydroxymethylfurfural(5-HMF)began to appear from 4-Z processed PCPs but was not observed in the previous steaming.With sugar as the variable,the processed PCPs were divided into 4 groups by HCA and PCA methods.The 0-Z and 1-Z PCPs were group A,the 2-Z,3-Z and 4-Z PCPs were group B,the 5-Z,6-Z,7-Z,and 8-Z PCPs were group C,and the 9-Z PCPs were group D.Antioxidant experiments demonstrated that 4-Z PCPs had the strongest activity in free radical scavenging.Further MH7 A cell experiment showed that the activity of 4-Z PCPs was superior to 0-Z and 9-Z PCPs in terms of cell proliferation inhibition,cell migration,cell invasion and ELISA assay.The 4-Z PCPs were separated by anion exchange chromatography and divided into 3 components.Through an antioxidant experiment,the 0.1 mol/L Na Cl solution elution component(PCP-F0.1)was selected for further purification.The PCP-F1 with a molecular weight of37.46 k Da was purified from PCP-F0.1 by Sephadex G200 gel column chromatography.The PCP-F1 was composed of glucose,mannose,galactose,rhamnose and galacturonic acid with a molar ratio of 3.5:2.5:1.3:1.8:0.8.The main chain of PCP-F1 was →3)-α-DGlcp,→2)-α-D-Glcp(6→,→1)-?-D-Glcp(2→,→2)-α-D-Gal Ap(3,4→,→1)-?-DManp(3→,→2)-α-D-Glcp(3→,branched for →3)-α-D-Glcp,→2)-?-D-Galp(4→,→1)-?-D-Glcp(2→,→2,4)-α-D-Manp(6→,→3)-α-L-Rhap(4→.The results of antioxidant activity showed that PCP-F1 could scavenge the DPPH and hydroxyl radicals.Conclusions: Through the modern instrument analysis methods and using in vitro antioxidant and MH7 A cell anti-inflammatory experimental analysis,we have completed the scientific connotation of "Nine-Steam-Nine-Bask" processed P.cyrtonema.With the content of monosaccharides as variables,we concluded that the quality of the 4-Z PCPs was the best by HCA and PCA methods.In addition,the antioxidant and MH7 A cell experiment of 4-Z PCPs further confirmed this result.Furthermore,the PCP-F1 was obtained by further purification and separation of the chromatographic column from 4-Z PCPs.The structure of PCP-F1 was confirmed by infrared spectrum,molecular weight,monosaccharide composition,methylation analysis and NMR analysis.The backbone of PCP-F1 consisted of →3)-α-D-Glcp,→2)-α-D-Glcp(6→,→1)-?-D-Glcp(2→,→2)-α-D-Gal Ap(3,4→,→1)-?-D-Manp(3→,→2)-α-D-Glcp(3→ with side chains →3)-α-D-Glcp,→2)-?-D-Galp(4→,→1)-?-D-Glcp(2→,→2,4)-α-D-Manp(6→,→3)-α-LRhap(4→ branches located at O-3 position of →2)-α-D-Gal Ap(3,4→. |
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