Studies On The Preparation, Structure And Bioactivities Of A Polysaccharide And Its Sulfated Derivatives From Phellinus Ribis

Phellinus ribis,a kind of fungi,belongs to Phellinus genus,Hymenochaetaceae family.As a Chinese folk medicine,the hot water extract of Phellinus ribis has been used for treating hepatitis and enhancing immunity.A lot of fungi in Phellinus genus are medical resources,such as Phellinus pini,Phellinus rimosus,Phellinus igniarius, Phellinus linteus,Phellinus baumii and so on.It was reported that many polysaccharides obtained from these fungi had various activities,for example,the acidic polysaccharide isolated from Phellinus linteus enhanced cell-mediated immunity,and the protein-bound polysaccharide had a direct anti-tumor effect.The neutral polysaccharide from Phellinus linteus showed hypoglycemic and blood lipid lowering effects.Extract of Phellinus conchatus,Phellinus baumii and Phellinus rimosus showed cytotoxic,free radical scavenging and antihepatotoxic activity, respectively.No report on the active constituents of Phellinus ribis was found till now. This paper reported an immunomodulatory polysaccharide named PRP isolated and fractionated from Phellinus ribis.Its physicochemical properties were analyzed,and its structure was determined by chemical analysis and spectrum technology,indicating PRP was a novel glucan purified from Phellinus ribis for the first time.After PRP was modified by sulfation,effects of PRP and its sulfated derivatives on tumor cells and their immuno-modulating activities in vitro were studied.All the results were summarized as follows.(1)Optimization of extraction process of total Phellinus ribis polysaccharide (TPRP).Through orthogonal experiment,the optimum conditions of TPRP extraction were 90℃,four hours,three times and 15mL H₂O per gram Phellinus ribis. Fractionated Phellinus ribis polysaccharide(FPRP)was obtained by deproteinization using a combination of trypsin enzymolysis and Sevag method and ethanol precipitation fractionation.The total sugar content of FPRP was 87.08%and protein content was 7.83%determined by phenol-sulfuric acid method and Folin-phenol method.(2)Isolation and Purification of Phellinus ribis polysaccharide(PRP).An active polysaccharide fraction named as PRP was obtained from FPRP by DEAE-cellulose ion-exchange chromatography and Superdex 30 gel filtration chromatography.The homogeneity of PRP was verified by HPGPC and cellulose-acetafolic electrophoresis.PRP had a weight-average molecular weight (Mw)of about 8.59 kDa by HPGPC in reference to standard T-series Dextran.The total sugar content of PRP was determined to be 97.3%and protein content was 1.03%.The specific rotation of PRP was +16.8°(c 0.1,H₂O),indicating theβ-configuration of the sugar units.PRP had a positive response to phenol-sulfuric acid and anthrone-sulfuric acid,while had a negative response to carbazole-sulfuric acid, ninhydrin,I₂-KI and ferric chloride reaction.(3)Structure analysis of PRP.PRP was composed of only glucose monomer as detected by GLC of the alditol acetate derivatives of the components of PRP hydrolyzate.The periodate oxidation indicated PRP included(1→6)-linkage,(1→2) -linkage or(1→4)-linkage.After further Smith degradation of the periodate-oxidized PRP,the glycerol,erythritol and glucose were found with an approximate molar ratio of 5.0:3.0:1.0 by GLC after conversion to the corresponding alditol acetates.It was thus deduced that the molar ratio of(1→6)-linked(terminal),(1→4)-linked and (1→3)-linked glucosyl residues was 5.0:3.0:1.The fully methylated product of PRP was hydrolyzed with acid,converted into alditol acetates,and analyzed by GLC and GLC-MS.Four types of glucose derivatives in a relative molar ratio of 1.0:3.0:4.0:1.0 according to the peak areas were as follows:1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl -glucose(terminal Glcp),1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-glucose(1,4-Linked Glcp),1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-glucose(1,6-Linked Glcp),and 1,3,5,6 -tetra-O-acetyl-2,3-di-O-methyl-glucose(1,3,6-Linked Glcp).PRP had no absorption at 280 nm or 260 nm in the UV spectrum,indicating the absence of protein and nucleic acid.The FT-IR spectrum showed that PRP had the hydroxyl stretching vibration of polysaccharide and that the pyranose residues of PRP were linked inβ-configuration.Partial acid hydrolysis indicated PRP had a possible(1→4), (1→6)-linked backbone,and the terminal glucosyl residues resided in side chains.The data of ¹H NMR and ¹³C NMR indicated PRP had three kinds ofβ-glycosal linkages. The ¹H and ¹³C NMR chemical shifts of PRP were assigned according to 2D ¹H,¹H COSY,¹H,¹³C HMQC experiments.On the basis of the above-mentioned results,it can be concluded that PRP is composed of a repeating unit having the possible structure as:(4)Antitumor and immunological activities of PRP in vitro.PRP did not show inhibition effect on Bel-7402,HepG2 and MDA231 cells in vitro by MTT assay.PRP showed an obvious splenocyte stimulating effect at a dose of 20μg/mL,when the dose was higher or lower,the proliferation index decreased.PRP could co-stimulate spleen lymphocyte proliferation with ConA(2μg/mL)at a low dose of 5 or 10μg/mL.while could not at high doses of 50 and 100μg/mL.(5)Snlfation of PRP and structure analysis the sulfated derivatives. Chlorosulfonic acid-formamide reagent was used for the sulfation of PRP.Four derivatives with different degrees of sulfation,PRP-Sâ… ,PRP-Sâ…¡,PRP-Sâ…¢and PRP-Sâ…£,were obtained through altering the ratio of the PRP and the reagent.The sulfate contents of PRP-Sâ… ,PRP-Sâ…¡,PRP-Sâ…¢and PRP-Sâ…£were 28.87%,51.52%, 60.41%and 56.03%respectively,the(?)ws of them were 14.61,20.54,22.43 and 21.42 kDa respectively.The degrees of optical rotation were +9.87°,+2.63°, -3.42°and -2.46°(c 0.1,H₂O),respectively.In the FT-IR spectrum of the sulfated derivatives,the presence of characteristic absorptions at 1258 and 810 cm^-1were conspicuous,which corresponds to the asymmetric S=O stretching vibration and the symmetrical C-O-S vibration.From PRP-Sâ… to PRP-Sâ…¢,the degree of sulfation was increasing as shown in ¹³C NMR spectrum.Sulfate groups were linked to C6-OH of branched glucosyl and(1→4)-glucosyl residues firstly.With the increase of sulfation degree,other hydroxyl groups would be sustituted.(6)Biological activities of PRP and its sulfated derivatives.The effects of PRP and its sulfated derivatives on tumor cells were determined with MTT assay.The results showed that the inhibition effect of sulfated derivatives on tumor cells increased with the increase of sulfation degree and the highest inhibition rate was 24.16%at the dose of 500μg/mL.The immuno-stimulating assay in vitro showed that PRP and its sulfated derivatives could stimulate spleen lymphocyte proliferation.The effect of PRP-Sâ… was in a concentration-dependent manner at the concentration range of 10～500μg/mL and proliferation index was 2.50 at 500μg/mL.While PRP, PRP-Sâ…¡,PRP-Sâ…¢and PRP-Sâ…£had optimal concentrations at 20,250,100, 100μg/mL respectively.With the degree of sulfation increase,proliferation effect of sulfated derivatives on spleen lymphocyte increased in a total tendency.PRP had no obvious effect on ECV304 cells,whereas sulfated derivatives showed a stronger inhibition activity.PRP could inhibit angiogenesis of zebrafish,sulfated derivatives could inhibit too,but the activities were lower than PRP.Originality of the article are the following:(1)A homogeneous polysaccharide named as PRP was obtained from Phellinus ribis for the first time.PRP had a(?)w of about 8.59 kDa and[α]_D²⁰+16.8°(c 0.1, H₂O).The total sugar content of PRP was 97.3%and the protein content was 1.03%.(2)The structure of PRP was determined as a newβ-D-glucan,which has a (1→4),(1→6)-linked backbone,with branches at the C-3 position of(1→6)-linked glucosyl residues.(3)Antitumor and immunological activities of PRP were studied for the first time. The results showed that PRP had an obvious immunostimulating activity with no cytotoxic activity.Zebrafish model experiment indicated that PRP could inhibit angiogenesis of zebrafish obviously.(4)Sulfation modification of PRP and the biological activities of the sulfated derivatives were studied for the first time.Results showed that antitumor activities, lymphocyte proliferation effects and inhibitions of endothelial cell were enhanced, whereas the inhibition activities of angiogenesis on zebrafish decreased after PRP was modified by sulfation.