| Ganoderma is one of the traditional fungi using as medicine and food in China. Itcontains a wide variety of bioactive components, such as triterpenes, polysaccharides,proteins, alkaloids, adenosines. And it has many significant biological abilities includinganti-tumor, immuno-enhancing, and anti-aging and so on. So, research and development ofGanoderma have attracted more attention in recent years. Now, there are many kinds ofGanoderma products at the market, but it’s hard to say whether their quality are good or bad,and it’s also difficult to identify whether the raw materials are authentic or adulterant. How toidentify authenticity and control the quality of different Ganoderma and their products havebecome the urgent problems. Ganoderma polysaccharides, as the main active substance ofGanoderma, had now become the main research object for testing and quality control ofGanoderma products. In our subject, polysaccharides from different Ganoderma fruitingbodies were extracted by suitable methods and partly hydrolyzed. Then differentchromatographic analysis methods were applied to build the fingerprint profiles ofpolysaccharides combined with chemometrics methods for characterization and identificationof polysaccharides. It can provide the reference and basis for inspection and quality control ofGanoderma products, and lay the foundation for further exploring the chromatogram-functionrelationship.In order to obtain real characterization of Ganoderma lucidum (G. lucidum)polysaccharides, G. lucidum fruiting bodies were chosen as experiment material to optimizethe best extraction condition. Under the optimal condition, not only the extraction efficiencywas higher, but also it had very little influence on the structure of polysaccharides, that is, theoriginal features of polysaccharides could be kept utmostly. The yield of polysaccharide wastaken as the index to investigate extraction conditions of different extraction methods basedon the single factor experiments and orthogonal experiments, and the best extractionconditions were determined. At the same time, by comparing the content of the fraction ofpolysaccharides of which molecular weight is more than10kDa, the influence of differentextraction conditions on molecular weight distribution of polysaccharides was investigated.The results showed that four extraction methods have effects on molecular weight distributionof G. lucidum polysaccharides, and the contents of the fraction of G. lucidum polysaccharideswith molecular weight more than10kDa by accelerated solvent extraction, ultrasonic-assistedextraction and water bath reflux extraction were similar, that is, the destruction of G. lucidumpolysaccharides were slight. But the structure destruction of G. lucidum polysaccharides withmicrowave-assisted extraction was relatively large. Ultimately we chose ultrasonic-assistedextraction and extraction conditions: extraction was at70℃for50min, and solid to liquidratio was1:20.Polysaccharides of37Ganoderma samples were extracted under the aboveultrasonic-assisted extraction conditions, and then hydrolyzed partly. The hydrolyzates weresubjected to pre-column derivatization with1-phenyl-3-methyl-5-pyrazolone (PMP) andRP-HPLC analysis. The chromatographic data were imported to professional software in order to obtain standard fingerprint profiles of G. lucidum fruiting bodies, G. sinense fruitingbodies and G. lucidum spores polysaccharides, and there are18,16and16commoncharacteristic peaks, respectively. By comparing samples with standard samples andHPLC-MS analysis, the common peaks were ten monosaccharide peaks and eightoligosaccharide peaks in G. lucidum fruiting bodies, nine monosaccharide peaks and sevenoligosaccharide peaks in G. sinense fruiting bodies, and nine monosaccharide peaks and sevenoligosaccharide peaks in G. lucidum spores. Then, similarity evaluation of different sampleswas performed. The results showed that the similarities between polysaccharides from thesame species and the same parts of Ganoderma were higher, but the similarities betweenpolysaccharides from the same parts but different species of Ganoderma were smaller. Thedata were further processed by hierarchical clustering analysis and principal componentanalysis. Polysaccharides from different parts or species of Ganoderma or polysaccharidesfrom the same parts of Ganoderma but from different geographical regions or different strainscould be differentiated clearly.To enhance the information of oligosaccharides in partial acid hydrolysates of G. lucidumpolysaccharides and characterize G. lucidum polysaccharides better, the oligosaccharides wereanalyzed on Sugar-Pak column. Taking the same individual glucose as external referencestandard, the data of monosaccharide on C18reverse phase column (usd for PMP-HPLCanalysis) and the data of oligosaccharide on Sugar-Pak Sugar column were integrated basedon the ratio of the two peak areas of individual glucose under two chromatographic conditions,and then the new data matrix were analyzed with chemometrics methods. The results showedthe data analysis based on the two column systems could reflect the characterization of G.lucidum polysaccharides, and the differences of polysaccharides from G. lucidum fruitingbodies and G. lucidum spores can be visually displayed by HCA and PCA. The results weresimilar with the results of fingerprint analysis by PMP-RPHPLC.Molecular weight distribution of polysaccharides extracted under the same extractionconditions were analyzed by HPSEC, and the chromatograms were segmented and the datawere processed by hierarchical clustering analysis. Results showed that molecular weightdistribution of polysaccharides of different G. lucidum were different. There were somedifferences between the different polysaccharides from the same parts of G. lucidum due to thedifferences of strains, geographical areas and cultivation methods and so on, and thedifferences could be distinguished with hierarchical clustering analysis. Polysaccharides fromdifferent parts of G. lucidum also had obvious differences, polysaccharides of G. lucidumfruiting bodies and mycelium can be clearly classified by hierarchical clustering analysis ofthe molecular weight data. For most of polysaccharides of G. lucidum fruiting bodies, thefraction of polysaccharides of which molecular weight were between10kDa and100kDawere major, and most of G. lucidum spores polysaccharides contained the fractions ofpolysaccharides of which molecular weight were10kDa~100kDa,100kDa~1000kDa evenalso more than1000kDa respectively. However, molecular weight distribution ofpolysaccharides of G. lucidum mycelium mainly was from10kDa to100kDa. |
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