Study On Chromatographic Fingerprints Of Polysaccharides From Ganoderma Lucidum Spore And Its Profile-Effect Relationship Of Immunocompetence

As one of the traditional fungi for both edible and medicinal use, Ganoderma lucidum has a history of more than two thousand years in China. With in-depth studies of components in Ganoderma lucidum such as polysaccharides, triterpenoids, alkaloids, adenosines and sterols, its physiological activities arrest more and more attention. Ganoderma lucidum polysaccharides, an outstanding representative of physiologically active substances among them, have played its important role as Ganoderma lucidum have come to market in the form of health care products, pharmaceuticals and functional food. Ganoderma lucidum and its products have mushroomed, while the quality of these products on the market is hard to distinguish, as well as to tell a true from a false. As the complexity of the composition and structure of polysaccharides, there is still a lack of analysis methods and quality standards which could effectively characterize and identify the polysaccharides from Ganoderma lucidum with physiological activity. In order to resolve the problem, we took the broken Ganoderma lucidum spore powder as the experimental material, and extracted Ganoderma lucidum polysaccharides by ultrasound-assisted extraction method. In our study, two methods of TFA partial acidic hydrolysis coupled PMP pre-column derivatization reverse phase high performance liquid chromatography (RP-HPLC) and acid-enzyme partial hydrolysis coupled high-performance anion-exchange chromatography (HPAEC) respectively were developed for detecting and characterizing the compositive and structural differences of polysaccharides extracted from the Ganoderma lucidum spore of different sources, and established two kinds of chromatographic fingerprints of the polysaccharides. Furthermore, we investigated the promoting effect of the polysaccharides from Ganoderma lucidum spore on lymphocyte proliferation by means of MTT experiments, which were applied to research the profile-effect relationship of immunocompetence.To get better characterization of Ganoderma lucidum polysaccharides information through the fingerprints, the conditions of TFA partial hydrolysis were investigated and optimized. It was discussed that the effect about TFA concentration, hydrolysis temperature and hydrolysis time on the total peak area of the common peaks in the PMP-HPLC analysis. And the best TFA partial hydrolysis conditions were obtained: hydrolysis with 0.4 mol/L of TFA under 110℃for 70 min.Following the TFA partial hydrolysis under the conditions above, the fingerprints of Ganoderma lucidum spore polysaccharides was established by use of the software Similarity Evaluation Eystem for Chromatographic Fingerprint of TCM (2004A), with 19 common peaks, the total peak area of which accounts for more than 95% of all peak area. The results of fingerprints similarity analysis of 15 samples of Ganoderma lucidum spore polysaccharides indicated that the similarity between products from the same origin was much higher than 0.97, while it was lower but still greater than 0.80 when the products originated in different areas. And significant differences could be found from the polysaccharides samples which are not from Ganoderma lucidum spore but under the same way of analysis.Similarly, in order to get the better ion chromatographic fingerprints, gradient elution conditions were optimized. Under the optimal gradient elution conditions above, the conditions of acid-enzyme partial hydrolysis were also investigated and optimized. It was discussed that the effect about H2SO4 concentration, hydrolysis temperature and hydrolysis time on the two indicators-the total peak area of the common peaks of monosaccharides and oligosaccharides fractions respectively in the HPAEC analysis. And the best hydrolysis conditions were obtained: hydrolysis with 3 mol/L of H2SO4 under 90℃for 2 h before enzymolysis. Under these hydrolysis conditions, the fingerprints of monosaccharides and oligosaccharides of Ganoderma lucidum polysaccharides hydrolysates were established respectively.In order to investigate profile-effect relationship of Ganoderma lucidum spore polysaccharides, MTT experiments were selected to reflect the immunocompetence of the polysaccharides. The proliferation data as the MTT experiments results of 20 samples of Ganoderma lucidum spore polysaccharides were got, with the results of HPAEC analysis of these samples, a multiple linear regression equation was established by using SPSS 17.0.