Study On Chromatographic Fingerprint Of Polysaccharides From G. Lucidum Mycelia Baesd On Immunocompetence And Optimization Of Its Fermentation

G. lucidum has a long history, it was regarded as a good omen and it was named "Rui Zhi" in the Spring and Autumn Period. There were books which wrote the pharmaceutical applications of G. lucidum in the Han Dynasty. Among them the "Shen Nong’s herbal classic" recorded G. lucidum in most detail, it said "A person will be always young and light after eating G. lucidum for a long time and his life will be lasting as God". In recent years, scholars have done scientific studies with G. lucidum. As a main bioactive substance of G. lucidum, G. lucidum polysaccharide has become a hot topic of research. Those studies focused on the separation, structure analysis, biological activity, active mechanism as well as quality monitoring of G. lucidum polysaccharide.The HPLC fingerprints of polysaccharides from G. lucidum mycelia were constructed, the relationships between HPLC fingerprints of polysaccharides and polysaccharide fractions and their immune activities were studied in this paper. Also the effects of carbon source and nitrogen source from submerged fermentation on the HPLC fingerprints were studied preliminarily.This study would provide technical means for monitoring the quality of polysaccharide from G. lucidum mycelium, and it would guide the industrial fermentation of G. lucidum.11 kinds of G. lucidum mycelia which were producted by submerged fermentation were from Shandong, Anhui, Zhejiang, Jiangxi and Jiangsu as well as other areas. Firstly water bath reflux extraction was used to extract the polysaccharides from G. lucidum mycelia, then the crude polysaccharides of G. lucidum mycelia were handled by decolorization and removal of protein. As a result, the purities of 11 kinds of polysaccharides were ranging from 77% to 86%. Secondly, the monosaccharide compositions of polysaccharides were analyzed, there were different monosaccharide types and mole ratios in the 11 kinds of polysaccharides, however, all of the polysaccharides contained Man, Rha, Glc, Gal, Xyl, Ara, Fuc as well as a little uronic acid. The results of IR and 13 C NMR showed that the anomeric carbon of the fourth polysaccharide sample was β bond configuration. The ranges of molecular weight distribution of polysaccharides from G. lucidum mycelia were wide, ranging from thousands to million, however, there was few molecular weight distribution in millions.The standard RP-HPLC fingerprint of polysaccharides from G. lucidum mycelia after partial acid hydrolysis and PMP derivatization was established. There were 17 common peaks and there were 8 monosaccharide peaks among them, the areas of common peaks were above 98.7% of the total area. The similarities between 10 kinds of pure polysaccharides and standard fingerprint were above 0.953, while the similarity of the fourth sample which contained the polysaccharides of fermentation liquid was 0.874. At last, the standard fingerprints of polysaccharides from G. lucidum mycelia, G. lucidum fruiting bodies and G. lucidum spores were compared, the similarity between G. lucidum mycelia and spores was 0.590, while the similarity between G. lucidum mycelia and G. lucidum fruiting bodies was 0.968. It indicated that the method of RP-HPLC can distinguish G. lucidum mycelia and G. lucidum spores; however, it couldn’t distinguish G. lucidum mycelia and G. lucidum fruiting bodies.The molecular weight distributions of polysaccharides in interval 50 k Da~100 k Da, 100 k Da~500 k Da, 500 k Da~1×103 k Da and >1×103 k Da were positively correlated with the proliferation of spleen cells. According to the regression coefficient, the immune activity of polysaccharides from G. lucidum mycelia mainly depended on the two high molecular weight ranges(500 k Da~1×103 k Da and >1×103 k Da). Peak 2, peak 3, peak 5, peak 6, peak 9 and monosaccharide peak such as Xyl, Rha, Gal, Ara and Fuc in RP-HPLC fingerprints had positive correlation with the proliferation of spleen cells.After the separation of the fourth polysaccharide by DEAE Sepharose Fast Flow and Sepharose CL-6B Gel column, two neutral polysaccharides named GLMNP1 and GLMNP2 were obtained as well as three acidic polysaccharides named GLMAP1, GLMNAP2 and GLMAP3. The molecular weights of five polysaccharide fractions was respectively 6.61×103 k Da, 5.01×103 k Da, 1.41 k Da, 9.42 k Da and 9.79 k Da. Moreover their purities were orderly 94.13%, 91.79%, 93.15%, 91.50% and 92.55%. The monosaccharide composition showed that all of the five polysaccharide fractions mainly consisted of Man、Glc N、Rha、Glc、Gal、Ara、Xyl and Fuc, however, there were different mole ratios with them.The result of analysis between molecular weight distribution and the proliferation of polysaccharide fractions showed that the polysaccharides in 50 k Da~100 k Da and 100 k Da~500 k Da were positively correlated with immune activity. There were 16 characteristic peaks in RP-HPLC fingerprints of polysaccharide fractions, peak 2, peak 6 and peak 7 of oligosaccharide peaks and Xyl, Rha, Gal, Ara and Fuc of monosaccharide peaks were totally positive correlation peaks. Common monosaccharide peaks such as Xyl, Rha, Ara and Fuc in RP-HPLC fingerprints of polysaccharides and its fractions were positive correlation peaks.Fermentation period affected the molecular weight distributions of polysaccharides. Day 6 to day 8 was the fastigium period to synthesis high molecular weight polysaccharide while the maximum molecular weight content of polysaccharide increased at the end of fermentation. Glucose, maltose or soluble starch as carbon source was favorable to the synthesis of high molecular weight polysaccharide. Maltose as carbon source was favorable to the synthesis of positive correlation peaks such as Xyl, Rha, Gal, Ara and Fuc according to the correlation peak index. Yeast extract and soybean cake powder as nitrogen source was good for improving the proportion of high molecular weight range. The polysaccharides had less species of monosaccharides in inorganic nitrogen culture while more species of monosaccharides and positive correlation peaks in organic nitrogen culture.Above all, HPLC fingerprints of polysaccharides from G. lucidum mycelia can be used for adulteration identification, while the fingerprint-pharmacodynamics relationship can guide the optimization of G. lucidum submerged fermentation in order to improve the quality of polysaccharides.