Research On Isolation, Purification, Structural Identification And Immunostimulating Activities Of Polysaccharides From Dendrobium Fimbriatum Hook

Dendrobium fimbriatum Hook.(Dendrobium SW.) is a kind of perennialepiphytic herb embodied in Pharmacopoeia of People’s Republic of China (2010Edition). In this paper, isolation, purification, structural identification andimmunostimulating activities of polysaccharides of D. fimbriatum were studied.Response surface method (RSM) was performed to optimize the extractionconditions of water-soluble polysaccharides of D. fimbriatum (WDFP). Resultsshowed that the highest extraction yield of9.68%was obtained under the optimalcondition:82.0°C of extraction temperature,51.0min of extraction time and33.3:1of the ratio of water to raw materials. The residues were further extracted with0.5mol/L NaOH and0.5mol/L HCl in turn, giving the base-solublepolysaccharides (BDFP) and acid-soluble polysaccharides (ADFP). The extractionyields of BDFP and ADFP were determined as1.01%and0.58%, respectively.WDFP was fractionated by ion-exchange chromatography of DEAE-Cellulose andfive fractions of WDFP-1，WDFP-2，WDFP-3，WDFP-4and WDFP-5were obtained.WDFP-1was further purified by gel permeation chromatography of Sephacryl S200and Sephadex G100, giving three homogeneous polysaccharides of WDFP-1a,WDFP-1b and WDFP-1c with the average molecular weight (Mw) of2.4×10~4Da,1.5×10~4Da and7.6×10~3Da, respectively. Similarly, one homogeneouspolysaccharide WDFP-4a with Mw of2.3×10~6Da and WDFP-5a with Mw of1.9×106Da was isolated from WDFP-4and WDFP-5by gel permeation chromatographyof Sephacryl S400, respectively.The structures of polysaccharides were characterized by methylation analysis,IR, GC, GC-MS and NMR.(1) WDFP-1a was composed of glucose and mannosewith the molar ratio of2.41:1.00. Methylation analysis indicated that WDFP-1ahad the backbone consisting of1,4-linked β-D-Manp and1,4-linked β-D-Glcp. TheO-2and O-3of1,4-linked β-D-Manp were substituted with acetyl groups.(2)WDFP-1b consisted of glucose and mannose. With respect to the glycosidic bonds,four forms including1,4-linked Glcp,1,6-linked Manp,1,3,6-linked Manp and1-linked Glcp were observed in WDFP-1b with the molar ratio of3.99:2.81:1.08:1.00.(3) WDFP-1c was also composed of glucose and mannose. There were four glycosidic bonds of1,4-linked Glcp,1,6-linked Manp,1,3,6-linked Manp and1-linked Glcp in WDFP-1c, corresponding to the molar ratio of4.05:3.19:1.13:1.00.(4) WDFP-4a consisted of galactose, glucose and mannose. There were fourtypes of glycosidic bonds of1,6-linked Galp,1,4-linked Glcp,1,3,6-linked Manpand1-linked Glcp with the molar ratio of9.28:5.41:1.03:1.00in WDFP-4a.(5)WDFP-5a consisted of galactose, glucose and mannose. With respect to theglycosidic bonds, five forms including1,6-linked Galp,1,4-linked Glcp,1,3,6-linked Manp,1,3,6-linked Galp and1-linked Glcp were observed inWDFP-5a with the molar ratio of15.53:7.71:1.72:1.00:2.63.Results of immunostimulating tests showed that polysaccharides of D.fimbriatum had high intestinal immune activities. Significant differences werefound between tested polysaccharide fractions. Compared to the other fractions,WDFP, WDFP-1and WDFP-1b showed higher positive effects.