| Ganoderma lucidum has been used to treat a variety of diseases including hepatitis, chronic bronchitis, asthma, coronary heart disease, angina, high blood pressure, high blood lipid, neurasthenia, cancer, etc. as a tradition Chinese medicine for thousand years in China and Southeast Asian. However, the fungus used in the treatment of diseases to a large extent depended on its pharmacological effects recorded in ancient literature in past, in recent years, an increasing number of researchers put their attention on biological substances and mechanism how they work in vitro/in vivo. As an important extract, polysaccharide/glycoconjugate had attracted more and more attention in past few years. A large part of papers dealed with monosaccharide composition and few ones focused on polysaccharide/glycoconjugate structure elucidation in past decades due in analysis and technique lag. So, there was no more detail and useful structural data of polysaccharide/glycoconjugate provided for development of polysaccharide medicines and functional foods, as well as for expounding biological mechanism. To further verify the fugus Ganoderma lucidum pharmacological activity and promote development of related products and drugs, together with enhancing depth and breadth of development of pharmaceutical products, the structures of polysaccharide/glyconjugate were studied herein, combination with chemical modification and pharmacological tests.1. Isolation, purification and physic-chemical characterization of polysaccharides/glycoconjugate from G lucidum fruiting bodiesTwo homogeneous polysaccharides (LZ-C-1and LZ-D-1) and five homogeneous polysaccharide-proteins/peptides (LZ-B-1, LZ-C-2, LZ-C-3, LZ-D-4and LZ-D-7) were purified from G. lucidum strain119fruiting bodies by successive steps including degreasing using ethanol, ultra-filteration, DEAE-Sepharose Fast Flow column, getting rid of free protein using Sevag method, Sephacryl-S or Sepharose CL-6B columns and HPLC. The relative molecular weight, total carbohydrate percentage and protein/peptide percentage were determined by HPLC, phenol-sulfuric acid method and BCATM kit, respectively, and the data were show as follows:LZ-B-1、 LZ-C-3and LZ-D-4were white powder samples. LZ-C-1and LZ-C-2were floc samples. LZ-D-7was white fibrous sample. LZ-B-1, LZ-C-1, LZ-C-2, LZ-C-3and LZ-D-1are soluble in water, DMSO. LZ-D-4and LZ-D-7can be dissolved in water, DMSO, and the same time the LZ-D-7had a smaller solubility than the LZ-D-4.2. Structures of polysaccharides/glycoconjugates2.1Primary structure elucidation of polysaccharide or polysaccharide moieties of glycoconjugates2.1.1Primary structure of polysaccharide moiety of polysaccharide-peptide LZ-B-1The primary structure of polysaccharide moiety of polysaccharide-peptide LZ-B-1mainly consisted of1,6-disubstituted-Galp,1,2,6-trisubstituted-Galp,1-substituted-Fucp,1,3-disubstituted-Glcp,1,4,6-trisubstituted-Glcp,1-substituted-Glcp in a molar ratio of ca. 1:4:1:0.5:0.5:0.5, along with a small amounts of1,6-disubsituted-Glcp,1-substituted-Galp,1-substituted-Manp and1,3,6-trisubstituted-Manp. So, the predicted repeating primary structure of polysaccharide moiety was as follows:2.1.2Primary structure of polysaccharide LZ-C-1The primary structure of polysaccharide LZ-C-1mainly consisted of1-substituted-Fucp,1,2,6-trisubstituted-Galp,1-substituted-Glcp,1,6-disubstituted-Galp,1,3-disubstituted-Glcp and1,4,6-trisubstituted-Glcp in a molar ratio of ca.1:1:2:4:2:2, along with a small amounts of1,6-disubstituted-Manp and so on. So, the predicted repeating primary structure of the polysaccharide was as follows:.2.1.3Primary structure of polysaccharide LZ-D-1The primary structure of polysaccharide LZ-D-1mainly consisted of1-substituted-Fucp,1,2,6-trisubstituted-Galp,1,6-disubstituted-Galp and1,6-disubstituted-Glcp in a molar ratio of ca.0.96:1.00:4.01:0.64, along with a small amounts of1,6-disubstituted-Manp,1,3-disubstituted-Glcp, and so on. So, the following predicted repeating primary structure may exist in the polysaccharide:Besides, residue1,6-disubstituted-Glcp was linked to residue1,2,6-trisubstituted-Galp. According to the relative area in1HNMR and the relative content by methylation analysis, it could be concluded that1,6-disubstituted-Glcp might be integrated in the repeating unit mentioned above via the1-position of residue B or other residues undetected in the NMR spectra every few repeating units.2.1.4Primary structure of polysaccharide moiety of polysaccharide-peptide LZ-D-4According to analysis by FT-IR, GC-MS and NMR and comparison with other polysaccharide structures mentioned above, the repeating primary structure of polysaccharide moiety of polysaccharide-peptide LZ-D-4might be the one of two structures as follows: 2.2Determination the polysaccharide-protein/peptide glycosylationβ-Elimination reaction revealed that the linkages between the polysaccharide moiety and protein/peptide moiety of polysaccharide-protein/peptide all might belong to N-glucosidic linkages.3Chemical modification of polysaccharide-peptide LZ-D-4The polysaccharide-peptide LZ-D-4isolated from G. lucidum by UF, gel filtration had a molecular weight of1.56×104Da, but its water-solubility was not very satisfactory. Its derivative, LZ-D-9, was prepared using sulfated agent (CSA:Pyr=1:6). The total yield of the sulfated product was approximately73%, and the content of sulfur was determined to be～2.30%, so the D.S. value was calculated to be0.12. The molecular weight was estimated to be1.30x104Da by HPLC. Another derivative, LZ-D-10, was acquired using Pyridine and Acetic anhydride as esterifying agent. The total yield of product was approximately90%, and the content of acetyl was determined to be7%and the D.S. value was calculated to be0.85. The molecular weight was estimated to be1.47x104Da. In our research, two formulae for calculating acetyl content and D.S. according to relative area in HNR were proposed as follows:1) Calculation for acetyl percentage 2) Calculation for D.S.D.S. refers to the average number of acetyl residues on each monosaccharide residue, which could be calculated as follows:4Pharmacological properties for samplesAll fractions were tested in vitro by BALB/c, L1210cell line and PC12cell line in our research.4.1Spleen cell proliferation assayIt was found that fraction GLPD had a better property to promote proliferation of mouse spleen lymphocytes in a dose-dependent manner than GLPB and GLPC after UF and total extract GLP. At the concentration of500μg/mL, the proliferation rate of MSLs was increased to197%(The proliferation rate of MSLs was increased to284%by treatment with positive control, PHA, at the concentration of10μg/mL.). Therefore, UF can play a certain effect to isolate some fractions which have a better property to stimulate the proliferation of MSLs. Samples GLPBW, GLPBS2, GLPDS1and GLPDS2isolated by DEAE-Sepharose Fast Flow column had better properties to stimulate the MSLs than other tested samples also isolated by DEAE-Sepharose Fast Flow column, the highest proliferation rate increased to273.05%,280.10%,282.34%and260.04%, respectively, which were close to the proliferation rate of PHA. Meanwhile, other samples had some properties to stimulate MSLs slightly. The proliferations of MSLs for fractions GLPB, GLPC and GLPD and their derivatives isolated by DEAE-Sepharose Fast Flow were investigated and results indicated that fractions GLPBW, GLPBS2, GLPCS1, GLPCS2, GLPDS1and GLPDS2had better properties to stimulate MSLs than their mother fractions. The proliferations of MSLs for all homogeneous were studies and data indicated that sample LZ-D-7had a strong property to stimulate MSLs. At the concentration of50μg/mL, the proliferation rate of MSLs was increased to283.58%, which was very near to that of PHA. At the concentration of200μg/mL, LZ-D-7had a better property than PHA. At the concentration of500μg/mL, the proliferation rate of MSLs was increased to333.15%. At the same time, samples LZ-B-1and LZ-D-1had a good property to stimulate MSLs proliferation to some extent. Other samples could slightly stimulate MSLs proliferation. The proliferations of MSLs for sulfated derivative LZ-D-9and acetylated derivative LZ-D-10were also investigated, and results indicated that only LZ-D-10had a slight higher bioactivity that LZ-D-4. So, it was thought that acetylation could enhance the proliferation of MSLs of polysaccharide to some extent.4.2Antitumor testThe experiment of inhibiting L1210cell line in vitro for all samples was done, and the results revealed that a large part of samples had no properties to inhibit L1210except for few samples for example GLPD, GLPB,LZ-D-4and LZ-D-9having slight bioactivities.4.3Rehabilitation study of PC12cell after treatment with H2O2by samplesAll samples were applied to investigate rehabilitation rate of PC12cell after treatment with H2O2, and the results indicated that samples GLPB, GLPB, GLPC,GLPD, GLPBS1, GLPCS1, GLPCS2and GLPDS1had properties to rehabilitate PC12cell in a dose-dependent manner, as the concentration increasing, the bioactivity was higher. At the same time, sample LZ-D-10exhibited a strong bioactivity to rehabilitate PC12cell, at the concentration of500μg/mL, the rehabilitation rate was145.46%, which was very close to that of positive control (NGF). The data also revealed that acetylation could improve rehabilitation rate of polysaccharide/glycoconjugate to PC12after treatment with H2O2. |
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