Isolation, Purification, Chemical Modification And Biological Activities Of Polysaccharides From The Fruiting Bodies Of Ganoderma Lucidum

Ganoderma lucidum (GL) is a kind of Eumycete which had played a important role in Chinese medicine and had been used for different diseases. It was reported that GL possessed various bioactivities, including immunostimulation, anti-tumor, hepatoprotective, antioxidation, anti-senility, anti-viral, anti-inflammatory and antidiabetics. Ganoderma lucidum polysaccharides (GLP) isolated from the fruiting body of Ganoderma lucidum has attracted more and more attention in recent years. Recent research has confirmed that the biological activities of polysaccharides are related to its structure, so structure modification of polysaccharide is an important method to produce new active polysaccharides. In this study polysaccharides from the fruiting bodies of Ganoderma lucidum were isolated, fractionated and purified, then these fractions and homogeneous polysaccharide was sulfated and their pharmacological function was tested. The results of this paper have extremely important scientific value to learn the rule of sulfated GLP and improving the bioactivity of polysaccharide of GL.This dissertation mainly study on three aspects:isolation and purification of polysaccharide of GL, derivatization and bioactivity of polysaccharides from the fruiting bodies of Ganoderma lucidum are discussed in detail on this paper.1. Isolation and purification of polysaccharides from the fruiting bodies of Ganoderma lucidum1.1Isolation, fractionation and purification of GLBy different methods17fractions or homogeneous polysaccharides was obtained. Fractions G1, G2, G3was fractionated according their molecular weight. G3-1and G3-2form G3was obtained by ethanol precipitation method. Alkali and water soluble fractions Al, A2and Alkali soluble and water insoluble fraction A3was produced by alkali extract method. LZ1, LZ2, LZ4, LZ5and LZ6was fractionated by the anion exchange column. LZ5-W, LZ5-W-2, LZ5-S and LZ5-W-2-A was got by the affinity column and gel filtration column. LZ5-S and LZ5-W-2-A was homogeneous polysaccharides by identification of HPLC. Molecular weight of LZ5-S and LZ5-W-2-A was1.35×104Da and4.09×10Da respectively. LZ5-W-2-A is white floe sample; LZ5-S is faint yellowish floc sample. They are both soluble in water and formamide. The monosaccharide components of LZ5-S, LZ5-W-2-A were identified by HPAEC. LZ5-S consisted of galactose, glucose, mannose and GluA in molar ratio of2:4:1:2, it also contained arabinose and xylose; LZ5-W-2-A consisted of galactose, glucose, xylose and GluA in molar ratio of2:2:1:1.1.2Purification of LZ5-W by affinity chromatographyAffinity chromatography was used to purify LZ5-W. Firstly the concentration of eluant alpha-Methyl-D-mannopyranoside was investigated. The optimal concentration was0.05M. Further the load capacity of ConA-Sepharose4B for LZ5-W was studied. The results showed that the load capacity is above20mg/mL. LZ5-W-2was purified from LZ5-W by affinity chromatography; the ratio of mannose in LZ5-W is enhanced37.2%.1.3Decoloration of GL polysaccharides by macroporous resinBecause the color of GL polysaccharides influenced seriously the measure of sulfate Decoloration of GL polysaccharides was studied. Four kinds of macroporous resin T1, D303, D315, HZ806was selected to decolor the G3. The results showed that T1was the best. At the best sample concentration is2-3mg/mL, the decoloration rate is93.6%while the polysaccharide contain rate is32.75%. After decoloration there are no changes on sugar concentration, but the protein concentration decreased obviously. G1、G2、G3-1、G3-2、 LZ5、A1、A2polysaccharide fractions were decolored using the macroporous resin.2. Sulfated modification of active polysaccharideThe single factor experiment showed that the volume ratio of chlorosulfonic acid to pyridine, reaction temperature, reaction time largely effected the sulfation modification of the polysaccharides. By the results of L9(34) orthogonal test and the range analysis on DS and yield, the optimum sulfamic acid conditions were examined as follows:According to DS, the influence of different factors to sulfated substitute rate was the volume ratio of chlorosulfonic acid to pyridine>temperature>time. The best group was B2A2C3, it meaned the volume ratio of chlorosulfonic acid to pyridine was1:8; sulfation temperature was80℃; sulfation time was2h. According to yield, the influence of factor was temperature>time>the volume ratio of chlorosulfonic acid to pyridine. The best group was A1B3C3. It meaned the volume ratio of chlorosulfonic acid to pyridine wasl:10; sulfation temperature was60℃; sulfation time was3h.44kinds of sulfated was got according to the optimal sulfated method. The highest DS of the derivative is0.66. The yield range is40%～83.2%to different samples. Sulfate groups is hydrophilic groups, it can increase the water solubility of the sample obviously. A3can not insoluble in water, but sulfated A3is water soluble.3. Study on bioactivity of polysaccharides from the fruiting bodies of Ganoderma lucidum and their derivativesThe effect of all polysaccharide fractions and their sulfated derivatives on immunostimulation, antitumor, anticoagulation and antimicrobial were tested in vitro.3.1Effect on immunostimulationThe immunostimulation effect of all GLP fractions and their sulfated derivatives was investigated by stimulating the production of NO of RAW264.7macrophage cell line. It was found that Both of GLP and their derivatives activated RAW264.7to secrete NO in concentration-dependent manner. Then by stimulating proliferation of mouse spleen lymphocyte G1-S2of low DS is better than G1-S5of high DS. However G2-S5of high DS has better effect on stimulating the proliferation of MSLs than G2-S2of low DS. G3-2-S2of low DS is better than G3-2-S5of high DS. Homogeneous polysaccharide LZ5-S also stimulated obviously proliferation of MSLs, but LZ5-S2sulfated from LZ5-S had weaker effect on stimulating proliferation of MSLs than LZ5-S. The sulfated fraction A3-S1derived from A3obtained the function of on stimulating proliferation of MSLs and activating RAW264.7to secrete NO, which A3did not possess.3.2Effect on anticoagulationThe anticoagulant effect of Ganoderma lucidum extract on APTT is not obvious, whereas their derivatives have obvious effect on APTT at1.25mg/mL. A1, A2and the derivatives of G2have prominent anticoagulation on APTT. The sulfated fraction derived from A3obtained the function on anticoagulation which A3did not possess. With the increasing of DS, G1-S5of high DS had stronger effect on APTT than G1-S2of low DS. All the derivated samples had anticoagulation effect on APTT; however, there is no effect on TT and PT. It indicated GLP sulfated derivates exerted anticoagulation effect through endogenous coagulation system.3.3Effect on anti-tumorThe inhibition effect of G1, G2and G3on K562cell was not obvious. G1, G2showed a little effects on K562, whereas their sulfated derivates displayed slightly effect. LZ2, LZ4and their sulfated derivates also did not show inhibition effect on K562cell. Sulfated fraction LZ5-S showed a little better than LZ5on inhibition effect on K562cell. 3.4Effect on antimicrobialGanoderma lucidum Polysaccharide and sulfation derivates did not show effect of antimicrobial on Escherichia coli, Staphylococcus auresus and Bacullis subtilis in vitro at5mg/mL concentration.