| Dioscorea bulbifera L.(D. bulbifera) is a liana widely distributed throughout the tropics and temperate regions. The rhizome of D. bulbifera has been used as a traditional Chinese Medicine for over2000years to treat thyroid diseases, spasmodic, leprosy and tumors. Polysaccharides are considered as the active components of D. bulbifera. It has been reported that the polysaccharide extracts from D. bulbifera have many pharmaceutical activities. However, there is little information about D. bulbifera polysaccharides regarding their fractionation, structural characterization and biological functions. In this paper, we described the extraction, fractionation and the structural features of a neutral polysaccharide (DBPN) from D. bulbifera. Furthermore, the main fraction of DBPN, DBPN-UD, was isolated. Its sulfated and carboxymethylated derivatives were synthesized.The antitumor activities of DBPN-UD and its derivatives were evaluated. The water-soluble polysaccharides were extracted from the rhizome of D. bulbifera with hot water, precipitated by80%ethanol. The polysaccharide mixture, referred to DBP, was separated on a preparative DEAE-Cellulose column into four fractions: one neutral fraction (DBPN) and three acidic fractions (DBPA-1, DBPA-2and DBPA-3) corresponding to0.0,0.1,0.3and0.5M NaCl elution, respectively. The major structural features of D. bulbifera polysaccharide fractions were elucidated by using high performance liquid chromatography and enzymolysis. The results showed that DBPN was heterogeneous and contained galactan and glucan. DBPA-1might be composed of homogalacturonan (HG) and galactan domains. DBPA-2might contain both homogalacturonan (HG) and small amount of type-I rhamnogalacturonan (RG-I) domains and DBPA-3might be composed of homogalacturonan (HG) domain.DBPN was completely fractionated by stepwise ethanol precipitation, which produced six homogenous fractions: DBPN-1(26.0%), DBPN-2(1.8%), DBPN-3(3.0%), DBPN-4(2.0%), DBPN-5(3.2%), and DBPN-6(2.6%) corresponding to final ethanol concentrations of40%,45%,50%,55%.65%, and75%with molecular weight of22.4,1.0,0.73,0.54,0.36and0.26kDa, respectively. Their structural features were elucidated by high performance liquid chromatography, enzymolysis, Fourier transform infrared spectroscopy and13C-nuclear magnetic resonance spectroscopy. The results indicated that DBPN contained galactan and glucan. The galactan was a linear β-(1,4)-D-galactan without side chains and contained galactose (96.9%). The glucan was a starch-like molecule. This is the first report of a linear β-(1,4)-D-galactan without side chains from D. bulbifera. D. bulbifera would be a suitable source for linear β-1,4-D-galactan in good yield and by simple preparation method. Sulfation with SO3-pyridine yielded six DBPN-UD sulfates: S-DBPN-UD-0.1, S-DBPN-UD-0.25, S-DBPN-UD-0.5, S-DBPN-UD-1, S-DBPN-UD-2and S-DBPN-UD-4with DS of0.12,0.26,0.63,1.42,0.92and1.02；Carboxymethylation with sodium salt of monochloro acetic acid (SMCA) yielded six DBPN-UD carboxymethylates: CM-DBPN-UD-0.1, CM-DBPN-UD-0.25, CM-DBPN-UD-0.5, CM-DBPN-UD-1, CM-DBPN-UD-2and CM-DBPN-UD-3with DS of0.16、0.21、0.26、0.46、0.62and0.70.The anticancer activities of DBPN and its derivatives were determined using an MTT assay and three cancer cell lines including sarcoma180and human colon cancer cells HCT-116and HT-29.. The results showed that sulfation and carboxymethylation of DBPN enhanced its inhibitory activityin a DS dependent manner. DBPN-UD and its derivatives were also tested for their anti-galectin-3activities using a galectin-3-mediated hemagglutination assay. DBPN-UD showed potent inhibition to the agglutination with an MIC of12.5μg/ml, similar to lactose, a standard galectin-3inhibitor. Sulfation and carboxymethylation significantly decreased the anti-galectin-3activity of DBPN-UD. The discovery that DBPN-UD had anti-galectin-3activity is valuable in guiding the preparation of effective pharmaceuticals from D. bulbifera for cancer prevention and treatment.The results of this paper will provide the theoretical basis for further research and development of D. bulbifera polysaccharides and improve the application of D. bulbifera polysaccharides. |
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