Structural Characterization and Antioxidative Activity of Low-Molecular-Weights Beta-1,3-Glucan from the Residue of Extracted Ganoderma lucidum Fruiting Bodies

The major cell wall constituent of Ganoderma lucidum (G. lucidum) is β-1,3-glucan. This study examined the polysaccharide from the residues of alkaline-extracted fruiting bodies using high-performance anion-exchange chromatography (HPAEC), and it employed nuclear magnetic resonance (NMR) and mass spectrometry (MS) to confirm the structures. We have successfully isolated low-molecular-weight β-1,3-glucan (LMG), in high yields, from the waste residue of extracted fruiting bodies of G. lucidum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay evaluated the capability of LMG to suppress H2O2-induced cell death in RAW264.7 cells, identifying that LMG protected cells from H2O2-induced damage. LMG treatment decreased H2O2-induced intracellular reactive oxygen species (ROS) production. LMG also influenced sphingomyelinase (SMase) activity, stimulated by cell death to induce ceramide formation, and then increase cell ROS production. Estimation of the activities of neutral and acid SMases in vitro showed that LMG suppressed the activities of both neutral and acid SMases in a concentration-dependent manner. These results suggest that LMG, a water-soluble β-1,3-glucan recycled from extracted residue of G. lucidum, possesses antioxidant capability against H2O2-induced cell death by attenuating intracellular ROS and inhibiting SMase activity.

Beta-1,3-D-glucans in G. lucidum have high molecular weights and low solubility in neutral water solutions. This creates the need to degrade glucan into smaller sizes for further application. Polysaccharide extraction from G. lucidum produces a large amount of residue as waste material. Therefore, the development of a "green" method of production, which recycles used materials and low volumes of waste, is of high importance. The present study's methods for isolating β-1,3-D-glucan are efficient and low cost when compared with other extraction methods, providing highquality products from the residue of G. lucidum, which may be useful in commercial applications.
Oxidative stress is implicated in the production of superoxide radicals in biological systems. ROS released from 2 Journal of Biomedicine and Biotechnology phagocytic cells initiates a wide range of toxic oxidative reactions and excessive formation of ROS causes cell apoptosis. Administration of antioxidants, such as medicinal herbs, can protect cells by attenuating injury caused by oxidative stress [13]. Almost all cell types express sphingomyelinase (SMase). Intracellular ceramide production increases the hydrolytic activity of SMases [14]. Ceramide is an intracellular lipid mediator that responds to various stimuli including stress. The inflammatory oxidant, H 2 O 2 , induces rapid increases in ceramide levels because of hydrolysis of sphingomyelin (SM) in plasma membranes, which contributes to the induction of various types of cell death. This study demonstrates that β-1,3-glucan activity is capable of interfering with SMases, and identifies β-1,3-glucan deriving from G. lucidum as a potential antioxidant source.
This study expanded the application of G. lucidum, reusing the waste materials from the extracted residue of fruiting bodies to obtain high-purity β-1,3-glucan in high yields. We also explored the biofunctions of low-molecularweight-G. lucidum β-1,3-glucan; an efficient antioxidative agent that eliminates ROS in H 2 O 2 -treated cells by interfering with SMases. Results indicated that low-molecular-weight β-1,3-glucan has potential future use as a biomedicine. The technology used during polysaccharide degradation might also have potential application on other medicinal herbs to release various glycans for biological assay.

Instruments in Structural Determination of β-1,3-
Glucan. High-performance anion-exchange chromatography (HPAEC) was used for the composition analysis of β-1,3-glucans. Glucose released by the acidic hydrolysis of LMG (4 N TFA 113 • C 6 h) and characterized using HPAEC-PAD with known monosaccharides as standard, a Dionex BioLC system (Dionex, Sunnyvale, CA, USA) with a CarboPac PA10 analytical column (4.5 mm × 250 mm) and a CarboPac PA10 guard column (4 mm × 50 mm) was used. Gas chromatography-mass spectrometer (GC-MS) was used for analysis of saccharide composition and linkages accord- ing to the methods described by Hakomori et al. [16].  [17]; Mass spectrometry was used for analyses of molecular weight and linkages. The molecular weights of the sodium adducts of oligosaccharides [M + Na] + were determined using MALDI-TOF MS (Applied Biosystems, Foster City, CA. USA). The glycan was mixed with 10 mg/mL of 2, 5dihydroxybenzoic acid (2,5-DHB) and 10 mM NaCl in the ratio of 5 : 5 : 3. In MS mode, the spectra were accumulated at an average of 1,000 to 2,000 shuts at a sample concentration of 1 mg/mL.

Cell Viability Assay.
MTT assay was conducted to assay cytotoxicity and cell viability, as described previously [18], dependent on the conversion of yellow tetrazolium salt to purple formazan product. Cells (1 × 10 5 cells/mL) were grown on a 96-well plate. Cells were treated with LMG (0-200 μg/mL) or LMG (0-150 μg/mL) combined with 200 μM H 2 O 2 for 24 h, after which an MTT solution (1 mg/mL in PBS) was added, and cells were incubated for 2 h. After the supernatant was discarded and DMSO was added to the well, the absorbance at 570 nm was measured using a spectrophotometer (Thermo Varioskan Flash, Vantaa, Finland).

Detection of Intracellular ROS Formation.
Levels of cellular oxidative stress were detected using the fluorescent probe, DCFDA, as described previously [19]. After being treated with 20 μM H 2 O 2 in the absence or presence of LMG (100 μg/mL), RAW264.7 cells were treated with 20 μM DCFDA for 30 min and then washed in PBS. DCFDA is mainly trapped in the cytoplasm and is oxidized to the highly fluorescent dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence intensities in cells were observed using fluorescence microscopy (Olympus IX71, Tokyo, Japan).

Suppression of Neutral and Acid SMase Activities by LMG.
Neutral SMase (nSMase) activity was estimated using an Amplex Red SMase assay kit (Invitrogen), as described previously [20]. Briefly, the assay mixture contained the following components in a total volume of 200 μL. The enzyme source (0.04 U/mL SMase, 1 U/mL HRP, 0.2 U/mL choline oxidase, 8 U/mL alkaline phosphatase, and 500 μM SM at pH 7.4) in the absence or presence of LMG (0-200 μg/mL) was prepared. For the nSMase assay, assay mixtures were incubated at 37 • C for 30 min. Assay of acid SMase (aSMase) activity was performed at pH 5.0. The reaction mixture contained 0.4 U/mL SMase, LMG (0-200 μg/mL), and 500 μM SM in 100 μL of 50 mM sodium acetate, pH 5.0, and cell samples were incubated at 37 • C for 30 min. A 100 μL working solution (consisting of 100 μM Amplex Red, 1 U/mL HRP, 0.2 U/mL choline oxidase, and 8 U/mL alkaline phosphatase in 100 mM Tris-HCl, pH 8.0) was added, and cells were incubated at 37 • C for 30 min. Fluorescence was measured using a microplate reader (Thermo Varioskan Flash) with excitation at 545 nm and emission at 590 nm.

Isolation of β-1,3-Glucans from Extracted Residue of G.
Lucidum. This study's aim was to obtain LMG from the extracted residue of G. lucidum and to examine its antioxidative activities. Alkaline extracted the ground fruiting bodies of G. lucidum to release a water-soluble component (20%) and a water-insoluble residue (80%). The water-soluble component contained heteroglycan, glycoprotein and others, and earlier studies have reported its immunomodulating function [3,6,10]. The recycling of β-1,3-glucan from the residue of G. lucidum was of interest due to the abundance of β-1,3-glucan in the cell walls of fungi and wide application in medicinal food [1,2,4]. Hydrolysis of the waste material, a water-insoluble residue, by strong acidic degradation (2 M HCl, 50 • C, 4 h) obtained LMG as low molecular weight glucans. This process provided smaller sized beta-glucans by narrowing down the high-molecular-weight-polyglycans, with the hydrolyzed glucans becoming water soluble. Further purification of the hydrolysate using size-exclusion chromatography (Sephadex G-15) provided three fractions; named HMG, LMG, and OSG. The isolated process is shown in Figure 1. The LMG is also obtained from the hydrolysate by ethanol precipitation (EtOH/H 2 O = 5/1) or by ultrafiltration (UF) with a molecular cutoff 500 Da to remove low-molecular-weight-components for large-scale isolation. Structural analyses and biological assays used the prepared LMG following collection and lyophilization.

Molecular Weights of LMG as Determined Using NMR and MALDI-TOF MS.
Recently, investigators developed NMR DOSY as a useful tool for measurement of molecular weights, including those of polysaccharides [17,23]. Using the equation in the experimental section, the estimated molecular weight of LMG is 3979 Da. The signal at 4.8 ppm, log D = −9.0, is H 2 O with molecular weight = 18 ( Figure 3). The signal of anomeric protons on LMG merged in the HOD at 4.8 ppm. The convergent dots in the same horizontal, at log D = −10.0, indicated that LMG has a narrow range in terms of size. Excepting the HOD signal, no dots appeared in the parallel line in the DOSY spectrum indicating that protons in LMG are connected, and LMG is a homogeneous glucan with differences in narrow range sizes.  MALDI-TOF MS analysis confirmed the presence of β-1,3-glucan in LMG. Figure 4 shows the molecular mass of LMG with gaps as 162 Da (one hexose unit) between peaks to peaks. Use of 2,5-DHB as a matrix substantially enhanced polysaccharide signals in the MALDI mass spectrometry [24]. This study obtained positive ions of glucan with unambiguous signals, observing LMG as sodiated ions ([Glcn + Na] + ) where n is the number of glucose units (6∼15). The mass region of ion peaks had a peak-to-peak mass difference of 162.1 Da, consistent with the repeating unit of the β-(1 → 3)-glucan. For example, when determining the degree of polymerization (DP) of LMG using MALDI-TOF, the average LMG mass appeared at DP = 9 (1499.1 Da, [Glc 9 + Na] + ) as a base peak. The molecular weights of LMG showed an average of 1,500 Da in MALDI-TOF MS, with slight differences from those obtained using the DOSY experiment. Based on these results, this study aimed to recover low molecular weight β-1,3-glucan from waste materials of G. lucidum residue. These recycled β-1,3-glucans might have potential commercial application as low-cost medicinal food.
Although some β-D-glucans are commercially available, they have different molecular weights, biological activities, and structures. Previous studies have identified the biological activities of β-1,3-glucan containing G. lucidum [5][6][7][8]. However, the antioxidative activities of low molecular weight glucans have yet to be fully understood. The structure-activity relationship (SAR) of LMG when exerting antioxidative effects was of interest. Using intracellular ceramide as a marker to determine the hydrolytic activity of SMases evaluated the antioxidative activity of isolated LMG, and also indicated if LMGs exerts protective effects on cells by attenuating injury under oxidative stress. Inflammatory oxidants cause this injury by inducing rapid increases in ceramide levels due to the hydrolysis of SM in plasma membranes. The study findings showed that LMG is capable of protecting cells against H 2 O 2 -induced apoptosis. LMG inhibited the activity of SMases to decrease the levels of intracellular ceramides, which may provide a useful indicator of the antioxidative activity of β-(1 → 3)-D-glucan.

Protective Effects of LMG against H 2 O 2 -Induced
Macrophage Death. Beta-(1 → 3)-D-glucan is the one of the major components of polysaccharide from G. lucidum, and has anti-inflammatory properties [5]. This study examined the antioxidant effects of LMG. MTT assay evaluated the cytotoxicity of LMG to RAW264.7 cells (Figure 5(a)), indicating that LMG is not meaningfully cytotoxic to RAW264.7 macrophages when LMG was added at 0-200 μg/mL for 24 h. Procedures also evaluated the protective ability of LMG against H 2 O 2 -induced injury. After 24 h H 2 O 2 (20 μM) treatment, MTT assay revealed 40% cell viability, which increased to over 80% after cotreatment of cells with LMG (100 μg/mL; Figure 5 (Figure 6(b)), the oxidized DCF fluorescent intensity was significantly greater than in untreated control RAW264.7 cells (Figure 6(a)). Cotreatment with LMG (100 μg/mL) provided markedly reduced 3.6. LMG Inhibited SMase Activity. In previous studies, factors of inflammation, including TNF-α, ROS, LPS, amyloidβ, IL-1β, and LDL, stimulated SMase activation activity, which then produced ceramide and initiated ceramide signal pathways to transfer death signaling [25][26][27]. This study evaluated the effects of LMG on bacterial SMase activity in vitro. Increasing LMG (0-200 μg/mL) reduced the activity of nSMase, with an IC 50 value of approximately 120 μg/mL (Figure 7(a)). LMG also had inhibitory effects on aSMase activity (Figure 7(b)). These data suggest that LMG has the potential to act as a SMase inhibitor. In Our previous research identified that a polysaccharide from Cordyceps mycelia possessed antioxidative ability and associated with SMase inhibitory effects, thus reducing levels of ceramides produced from hydrolysis of sphingomyelin [20]. Although findings did not fully elucidate the mechanism by which LMG exerts antioxidative effects, LMG's potential as a SMase inhibitor may be of importance for its future applications.

Conclusion
This study successfully isolated LMG, a low molecular weight β-1,3-glucan fraction, from the extracted residue of G. lucidum in high yields. A chemical method degraded the high-molecular-weight insoluble cell wall glucans into smaller sized compounds. This study also investigated the antioxidant activity of isolated LMG β-1,3-glucan to indicate the potential commercial value of G. lucidum glucans. LMG can protect cells by attenuating injury under oxidative stress caused by inflammatory oxidants. The study finding show that LMG, a water-soluble β-1,3-glucan with low molecular weight, recycled from extracted residue of G. lucidum, has activity against H 2 O 2 -induced apoptosis. LMG, therefore, is capable of exerting antioxidant effects by inhibiting SMases. The recycling of β-1,3-glucan from the remains of fruiting bodies could potentially provide a food supplement source of antioxidants for maintenance of body health. This is the first study to report the antioxidative effects of low molecular weight β-1,3-glucan from G. lucidum. The methods used have potential application in the isolation of antioxidative polysaccharides from other medicinal herbs.