Discussion and Analysis of Flammulina velutipes Polysaccharides Compositions , Molecular Weight and Monosaccharide Composition

The aim of the present work was to explore the composition, molecular weight and monosaccharide composition of Flammulina velutipes polysaccharides by DEAE Cellulose-52, High Performance Gel Filtration Chromatography (HPGFC) and HPLC with PMP derivative with the FVP isolated and purified from Flammulina velutipes. The results showed that Flammulina velutipes polysaccharides were composed of three kinds of polysaccharides; the molecular weight of FVP was composed of 4191338, 372779, 19002; the molecular weight of 372779, 19002, one of the biggest average molecular weight polysaccharides is neutral polysaccharide, the rest of the two kinds of polysaccharide composition of less average molecular weight is acidic polysaccharides and the proportion of two kind of acidic polysaccharides significantly greater than neutral polysaccharide, 44.31 and 37.76% respectively, only 17.93% of neutral polysaccharide. FVP was composed of Glc, Man, Gal, Xyl, Fuc; the highest content of Glc, followed by Gal and Man, Xyl and Fuc content is relatively low. The molar ratio was relatively 13.05:2.75:3.16:1.48:1.00. In conclusion, FVP contained the more Glc, the less Fuc and a small amount of galacturonic acid and glucuronic acid, which showed FVP may contain acidic polysaccharides.


INTRODUCTION
Polysaccharide is one of the important forms of naturally occurring sugar.It usually consists of more than 10 monosaccharide bases which are connected by glycosidic bond (Abdel-Akher et al., 1952;Agrawal, 1992;Pang et al., 2007), including polysaccharides (Ikekawa et al., 1982), storage structure and biological activity of polysaccharide (Mallavadhani et al., 2006;Pang et al., 2007).Because bioactive polysaccharide has much important biological activity, it has been applied in many areas such as functional food, biological medicine, biological materials (Wasser, 2002;Zhang et al., 2007).Bioactive polysaccharide is usually composed of several hundred to several thousand monosaccharides polymerization, whose nature is completely different from monosaccharides and biological activity of polysaccharides are related to its monosaccharide composition, molecular weight and structure (Yan et al., 2004).Flammulina velutipes Polysaccharides (FVP) is the most abundant biological active compounds content of Flammulina velutipes, has the good immunity, antitumor, protecting liver and enhance memory (Leung et al., 1997;Zheng et al., 2005;Peng et al., 2005).
The objective of this study was to improve the efficient use of FVP and the Flammulina velutipes postpartum added value and supply theoretical basis by adopting reasonable technical means to analyze the monosaccharide composition, molecular weight and component.

Isolation and purification of polysaccharide fractions:
In the present work, the extraction and isolation of FVP was performanced by boiling-water decoction and ethanol precipitation to yield crude polysaccharide (Chauveau et al., 1996;Yalin et al., 2006;York et al., 1986).The dried Flammulina velutipes were crushed into fine particles, extracted with 25 times of double-distilled water and centrifuged.The combined aqueous extracts were concentrated in a beaker for leaching by centrifugation at 4,500r/min for 20min.The supernate was concentrated in a rotary evaporator under reduced pressure.The concentrated solution was mixed with 80% alcohol followed by centrifugation at 4,500r/min for 20min.Then the Sevag method was used to remove protein components after re-dissolution of the crude polysaccharides (Bhandari et al., 1990;Miyazaki and Nishijima, 1982).The solution was then dialyzed against distilled water for 2 days and precipitated by adding ethanol until the concentration of ethanol reached 80%.The precipitate was collected by centrifugation and washed successively with absolute ethanol and acetone to give a light yellow powder.

DEAE cellulose-52 column chromatography analysis:
For additional purification the FVP were subjected to DEAE-52 cellulose column chromatography, eluting with distilled water (Angyal et al., 1974).Each 50 mL eluted fraction was collected and the content of sugar was monitored using the phenol-sulfuric acid method (Liu et al., 2007;Zhang et al., 2001).Fractions 8∼14 were combined, concentrated to 100 mL and designated as the APS fraction.Then, this material was subjected to Sephadex G-150 column chromatography (2×60cm), eluting with distilled water collecting 50 mL fractions with monitoring by the phenol-sulfuric acid method.
HPGFC analysis: Chromatographic condition.PMP-HPLC analysis: PMP-HPLC was carried out on an Agilent-1100 HPLC system equipped with a quaternary gradient unit and a refractive index detector (RID).The analytical column used was a ZORBAX Eclipse XDB-C 18 (250×4.6mm i.d., 5µm) with 0.1 mol/L phosphate as the eluant at column temperature 30°C.The wavelength for RID detection was 250nm.Elution was carried out at a flow rate of1mL/min at 30°C.
The preparation of PMP-polysaccharide derivations was as follows (Fu and O'Neill, 1995;Fu and O'Neill, 1995;Li et al., 2003): Freeze-dried polysaccharides (2mg) were dissolved in 1mL 2 M TFA (trifluoroacetic acid).N 2 was bubbled through the solution for 30 s and then the ampoule was sealed.After hydrolysis for 6-8 h rat 110°C in an oven, the solution was cooled to room temperature.The collected peak elutes were dried via rotary evaporation to remove residual TFA at 50°C and then neutralized by 0.3 M NaOH to make 5 mL water sludge mixture.After that, 300 uL 0.5 mol/L PMP and NaOH were added and heated at 70 for 30 min.The solution was cooled to room temperature and neutralized with addition of 300 uL of 0.3 mol/L HCl.One milliliter chloroform was added to the mixture, shaken thoroughly, centrifuged and the super-natant was collected.Finally, the supernatant was filtered through 0.45-um filters to eliminate dust particles.

Elution curve of FVP:
As can be seen from the Fig. 1, elution with distilled water obtained a neutral polysaccharide; elution with 0.1~0.5 mol/L NaCl obtained an acidic polysaccharide and appeared two peaks.
Figure 2   A HPGFC method was used to determine the molecular weight (Casu, 1982).Figure 3 shows the chromatograms of three kinds of polysaccharide compositions.The differences of column chromatography elution process, pretreatment of polysaccharide and protein removal process can lead to the differences of polysaccharide components.Of course, the differences of Flammulina velutipes species and origins may also lead to the differences of polysaccharide components.2.

HPLC of monosacharides of FVP:
The chromatogram in Fig. 5 shows that using an HPLC method, FVP was composed of five monosaccharides-Man, Glc, Gal, Xyl and Fuc-in a molar ratio of 13.05:2.75:3.16:1.48:1.00.Table 3 shows that the retention time were 15.606, 31.450, 35.824, 38, 519, 46.821 and the corresponding peak area were 12.6717, 60.1374, 14.5636, 6.8099, 4.6069.The FVP composition of polysaccharides in Glc content is higher, the second is the Gal and Man and Fuc content is low.Thus infer, FVP mainly be consisted by dextran and galactose glycan, mannan, xylose chitosan, fucus chitosan and other components may be mixed in it.

CONCLUSION
According to DEAE Cellulose -52 column chromatography analysis, it shows that the FVP was composed of three polysaccharide components.HPGFC Analysis shows that the molecular weights were 4191338, 372779, 19002,.With heavy molecular weight as 4191338 polysaccharide was neutral polysaccharide, the rest of the two polysaccharides were acidic polysaccharides.Among them, two of the acidic polysaccharides were accounted for a higher proportion, 44.31 and 37.76% and only 17.93% neutral polysaccharide components.
FVP was composed of five monosaccharides-Man, Glc, Gal, Xyl and Fuc-in a molar ratio of 13.05:2.75:3.16:1.48:1.00.Can be concluded that FVP mainly be consisted by dextran and galactose glycan, mannan, xylose chitosan, fucus chitosan and other components may be mixed in it.

Fig. 5 :
Fig. 5: HPLC of monosacharides of FVP Figure 4 shows that different standard samples of monosaccharides in the chromatographic conditions can be clearly separated.Samples of the standard monosaccharides peak time are shown in Table

Table 1 :
Analytical results of the FVP by HPGFC Table1shows that the retention time

Table 2 :
The peak time of standard monosacharides