Article pubs.acs.org/JAFC
Isolation, Structure Characterization, and Immunomodulating Activity of a Hyperbranched Polysaccharide from the Fruiting Bodies of Ganoderma sinense Xiao-Qiang Han,†,#,∥ Ben Chung Lap Chan,†,∥ Cai-Xia Dong,§ Yin-Hua Yang,‡ Chun-Hay Ko,† Grace Gar-Lee Yue,† Dan Chen,⊗ Chun-Kwok Wong,†,⊥ Clara Bik-San Lau,† Peng-Fei Tu,*,# Pang-Chui Shaw,‡ Kwok-Pui Fung,† Ping-Chung Leung,† Wen-Luan Hsiao,§ and Quan-Bin Han*,†,§ †
State Key Laboratory of Phytochemistry and Plant Resources in West China, Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China § School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China # State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China ‡ School of Life Sciences and Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China ⊗ Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China ⊥ Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong SAR, China ABSTRACT: A polysaccharide (GSP-6B) with a molecular mass of 1.86 × 106 Da was isolated from the fruiting bodies of Ganoderma sinense. Chemical composition analysis, methylation analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were conducted to elucidate its structure. GSP-6B contains a backbone of (1→6)-linked-β-D-glucopyranosyl residues, bearing branches at the O-3 position of every two sugar residues along the backbone. The side chains contain (1→4)linked-β-D-glucopyranosyl residues, (1→3)-linked-β-D-glucopyranosyl residues, and nonreducing end β-D-glucopyranosyl residues. An in vitro immunomodulating activity assay revealed that GSP-6B could significantly induce the release of IL-1β and TNF-α in human peripheral blood mononuclear cell (PBMC) and showed no toxicity to either PBMC or a human macrophage cell line THP-1. GSP-6B could also activate dendritic cells (DC) by stimulating the secretion of IL-12 and IL-10 from DC. KEYWORDS: Ganoderma sinense, polysaccharide, immunological activity, cytokine, dendritic cell
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INTRODUCTION Lingzhi is one of the most well-known functional foods and has been popularly used in Asian countries for a long time. The general name of Ganoderma species was cited as early as 100 B.C. in China.1 Although this genus is a big family, now the name Lingzhi usually means a single species Ganoderma lucidum, which contains various phytochemicals, such as triterpenoids, steroids, alkaloids, nucleosides, proteins, and polysaccharides.1 Polysaccharides are believed to be the major active ingredient, showing antioxidant activities,2−4 immunomodulating activities,5,6 and antitumor activities.7−9 The major bioactive polysaccharide is 1,3-linked β-D-glucopyranosyl with 1−15 units of 1,6-linked β-D-glucopyranosyl side chains.1 Besides G. lucidum, Ganoderma sinense is another official species of Lingzhi in China. It also exhibits multiple pharmacological effects, such as immunomodulating, antitumor, hepatoprotecting, antioxidant, and cholesterol-lowering activities.1,10−13 Recent studies indicated that these two food materials might be quite different. Ganodermic acids, often reported as anticancer compounds, are major components of the ethanol extract of G. lucidum, but little or even none at all is found in G. sinense.1,14 It is suggested that significant difference © 2012 American Chemical Society
might also exist in their polysaccharide profile. Compared to G. lucidum, of which the polysaccharide components have been well studied in terms of chemical structures and bioactivities, little is known about the chemistry of purified polysaccharides from G. sinense. Because the polysaccharide crude extract of G. sinense showed significant immunomodulating activity,15−19 and the structural characterization is essential for demonstration of the pharmacological mechanism and quality control, we start an exploration of chemistry and immunological activities of purified polysaccharides of G. sinense. Herein we report the isolation, structure elucidation, and in vitro immunomodulating activity of a novel hyperbranched polysaccharide from the fruiting bodies of G. sinense.
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EXPERIMENTAL PROCEDURES
Materials and Chemicals. Dried fruiting bodies of G. sinense were purchased from a herbal store in Hong Kong and authenticated by Received: Revised: Accepted: Published: 4276
December 20, 2011 April 12, 2012 April 12, 2012 April 13, 2012 dx.doi.org/10.1021/jf205056u | J. Agric. Food Chem. 2012, 60, 4276−4281
Journal of Agricultural and Food Chemistry
Article
hydrolyzed, reduced, and acetylated.22 The partially methylated alditol acetates were analyzed by GC-MS. NMR Analysis. GSP-6B (30 mg) was dried in a vacuum over P2O5 for 72 h and then exchanged with deuterium by lyophilization with D2O three times. After that, the sample was put in a 5 mm NMR tube and dissolved in 1.0 mL of 99.96% D2O. All spectra were obtained at 298 K on a Bruker Avance 700 MHz NMR spectrometer equipped with a TCI cryoprobe. Tetramethylsilane (TMS) was used as external standard in the 13C NMR test, and D2O was used as internal standard in the 1H NMR test. Partial Hydrolysis. GSP-6B (20 mg) was partially hydrolyzed with 0.1 M TFA at 60 °C for 10 h. The hydrolysate was then dialyzed against distilled water for 24 h in a dialysis bag with a 7000 Da cutoff. The residues in the dialysis bag was collected and coded GSP-6B-in. Amino Acid Analysis. GSP-6B (10 mg) was hydrolyzed under vacuum at 110 °C in 6 M hydrochloric acid for 24 h. The hydrolysis products were determined with a Hitachi 835-50 amino acid analysis analyzer, with a group of standard amino acids as the markers. The content of each kind of amino acids was calculated using the standard curves. Measurement of Immunomodulating Activity. Effect of GSP-6B on Cytokine Secretion of Human Peripheral Blood Mononuclear Cell (PBMC) .23 The fresh buffy coat was diluted with phosphate-buffered saline at a ratio of 1:1. The diluted sample (20 mL) was put in a 50 mL centrifuge tube together with an equal volume of Ficoll−Plaque Plus solution. The tube was then centrifuged at 800g for 20 min at 18 °C. The supernatant was discarded, and the PBMCs were resuspended in 4 mL of RPMI 1640 medium plus 10% fetal bovine serum (FBS). The cell number was counted, and the viability of the cell was checked by trypan blue exclusion assay. The isolated PBMCs were seeded in a 96-well flat-bottom microplate and incubated with GSP-6B, LPS, or dextran (0.00003− 100 μg/mL). After 24 h of treatment, concentrations of IL-1β and TNF-α in culture supernatant were determined using ELISA kits (BD Pharmingen Corp.). Polyminxin B (PMB), which is a specific inhibitor of LPS, was added in the sample of GSP-6B to exclude the influence of LPS. XTT Proliferation Assay .24 The cytotoxicity of GSP-6B on PBMCs and a human macrophage cell line (THP-1) was determined by XTT proliferation assays (Roche) according to the manufacturer’s instructions. In brief, 2 × 105 THP-1 cells or PBMCs were seeded into flat 96-well plates. The cells were cultured in the presence of GSP6B, LPS, or dextran at different concentrations for 72 h. After the incubation period, 50 μL of XTT/phenazinemethosulfate solution (20 μM) was added to each well for a further 4 h of incubation at 37 °C. Finally, the colorimetric changes of each well were measured at a wavelength of 490 nm. The toxicity represents the ratio of the OD of a well in the presence of tested compounds with the OD of the drug-free control wells. A cellular viability of at least 90% was considered to indicate a nontoxic compound. Effect of GSP-6B on Production of IL-10 and IL-12 in Human Dendritic Cell (DC) .25 For generation of human monocyte derived dendritic cells (DC) from PBMC, monocytes were positively purified from PBMCs by attachment method. Cells were cultured at 2 × 106 cells/mL in RPMI/10% FBS medium supplemented with granulocyteGM-CSF (50 ng/mL) and IL-4 (40 ng/mL) for 6 days. The immature DC were then harvested and incubated for 2 days by adding GSP-6B, LPS, or dextran (0.00003−100 μg/mL). Supernatants were collected, and the concentrations of IL-10 and IL-12 were determined by ELISA. Statistical Analysis. All experiments were repeated at least three times. Results are presented as the mean ± the standard error of the mean (SEM). Comparison of the data was performed using the singlefactor ANOVA test. Significance was defined as a p value of
