Electronic Journal of Biology

Introduction

Mushrooms have been known for their edible, medicinal resources and antitumor substances for many years. The fungi belonging to the genus Ganoderma are popular medicinal mushrooms, widely used in China, Japan and Korea over the past two millennia [1,2]. The most frequently cited Ganoderma species used in research publications on the cultivation, chemical analysis, pharmacology and medicinal effects is the Ganoderma lucidum (G. lucidum), an edible medicinal mushroom commonly known as Reishi or Manentake (Japanese) or Lingzhi (Chinese) [3]. The incredible curative properties have won it the titles of ‘supernatural mushroom’, ‘magic mushroom’ and ‘plant of longevity or immortality’, produced not only in its native East Asian countries such as China, India, Japan, Korea, Taiwan, and Malaysia but also in the USA. G. lucidum has been reported to have many pharmacological effects including immune-modulating, anti-atherosclerotic, anti-inflammatory, analgesic, chemopreventive, anti-tumour, radioprotective, sleep-promoting, antibacterial, antiviral (including anti-HIV), hypolipidemic, anti-fibrotic, hepatoprotective, diabetic, antioxidative and radical-scavenging, antiaging, hypoglycemic, and antiulcer properties [4- 10]. Reishi mushroom has now become recognized as an alternative adjuvant in the treatment of leukaemia, carcinoma, hepatitis, and diabetes.

Polysaccharides, triterpenes, sterols and peptidoglycans are the major chemical constituents of Ganoderma lucidum, along with oleic acid, soluble proteins, amino acids, ergosterol peroxide (5,8-epidioxy-ergosta-6,22E-dien-3-ol), and the cerebrosides (4E’,8E)-N-D-2’–hydroxylstearoyl-1-O- β-D-glucopyranosyl-9-methyl-4-8-sphingadienine, and (4E’,8E)-N-D-2’-hydroxypamitoyl-1-O-β-Dglucopyranosyl- 9-methyl-4-8-sphingadienine and cyclo-octasulfur along with inorganic ions like Iron, Manganese, Germanium, Magnesium, Zinc, Copper, and Calcium [11-13]. The fruiting body of the Ganoderma lucidum is shown in Figure 1. G. lucidum spore cell wall contains a high amount of polysaccharides, which are natural macromolecular compounds with complex and versatile biological activities. The spores also contain choline, betaine, tetracosanoic acid, stearic acid, palmitic acid, ergosta-7, 2, 2-dien-3-ol, nonadecanoic acid, behenic acid, tetracosane, hentriacontane, ergosterol, and β-sitosterol. One of the lipids isolated from G. lucidum is pyrophosphatidic acid [14,15].

Figure 1: Ganoderma lucidum (Reishi or Lingzhi).

In recent years, polysaccharides extracted from G. lucidum have been regarded as an important class of anticoagulants, immunomodulating and antitumour with antioxidant activities, antiproliferative activities, antiviral and antiprotozoal activities [16- 20]. G. lucidum polysaccharides such as β-Dglucans, heteropolysaccharides, and glycoprotein have been isolated and characterized; considered as the major contributors of bioactivity of the mushroom. β-D-glucans consist of a linear backbone of β-(1→3)- linked D-glucopyranosyl groups with varying degrees of branching from the C6 position. In addition to water-soluble β-D-glucans, β-D-glucans co-exist with hetero-polysaccharide chains of xylose, mannose, galactose, uronic acid and β-D-glucans–protein complexes that are present at 10–50% in dry G. lucidum, presence of various reactive groups in their structure, polysaccharides can be easily modified chemically and biochemically. Moreover, the presence of hydrophilic groups in their structure, such as hydroxyl, carboxyl and amino groups, enhance bio-adhesion with biological tissues, like epithelia and mucous membranes, forming non-covalent bonds, a useful strategy to improve the bioavailability of drugs included in drug delivery systems.

Extraction of polysaccharides

Polysaccharides extraction is fairly time consuming and slow process, the literature suggests several published articles using different approaches for the extraction of polysaccharides from the spores of G. lucidum. The most common approaches were hot water extraction (HWE) for the extraction of watersoluble polysaccharides and alkaline extraction is used for the extraction of water-insoluble polysaccharides.

Traditional use of hot water extraction (HWE) was the cause for a lower yield, longer extraction times and high-temperature process. To get the better yields other techniques like Ultrasound Microwave- Assisted Extraction (UMAE) by Sheng et al, Ultrasonic Assisted Extraction (UAE) by Liyan et al, [23] breaking the spores of the fungus G. lucidum by supercritical CO₂ by Yu-Jie et al, breaking the spores of G. lucidum by fermentation with Lactobacillus plantarum by Chaiyavat, Chakkrapong and Sasithorn [24] and alkaline extraction of polysaccharides (AEP) by Gao et al, [25,27] were reported. The percentage yields of different techniques were given in Table 1. The most effective procedure of AEP results showed optimized yields from the fruiting body of G. lucidum of 6.81% under alkaline extraction conditions.

Table 1. The percentage yield of polysaccharides extracted from Ganoderma lucidum using different

Extraction Procedure

The dried sample was grounded into a fine powder and defatted with petroleum ether, ethyl acetate and methanol. Then mixed with 80% ethanol and shaken at 30°C for 24 h, to remove most of the polyphenols and monosaccharides. Water-soluble polysaccharides were extracted stepwise with a 0.2 M phosphate buffer solution (PBS) of pH-7 at 25°C, 80°C and 120°C. In each step, the PBS suspension was centrifuged and to the supernatant was added a large quantity of ethanol to precipitate the polysaccharide. The precipitates of G. lucidum polysaccharide (GLP) were designated GLP I, GLP II and GLP III in the order of increasing extraction temperature.

The residue obtained from the last PBS suspension was then treated with 1% ammonium oxalate and acetic acid was added to the supernatant to precipitate the water-insoluble polysaccharides and was designated as GLPIV the residue obtained was treated with 5% NaOH at 25°C, acetic acid was added to precipitate the polymers and designated as GLPV. Ethanol was added to the supernatant to get the final polysaccharides GLPVI. Crude polysaccharides of G. lucidum at different stages of extraction were obtained. These extraction procedures were similar to those reported by Sone et al, [28-35] for Reishi mushroom and by Wang et al, [36] for G. tsuage mushroom. The procedure for the complete extraction process of polysaccharides from G. lucidum is depicted in Figure 2.

Figure 2: The complete extraction process of polysaccharides from Ganoderma lucidum [37].

Purification of Polysaccharides

Extracted polysaccharides were purified by a combination of techniques, such as ethanol precipitation, fractional precipitation, and acidic precipitation with acetic acid, ion-exchange chromatography, gel filtration, and affinity chromatography. The ethanol precipitation excludes the impurities from the polysaccharides. The separation of acidic and neutral polysaccharides can be achieved by anion-exchange chromatography on diethyl-amino-ethanol cellulose (DEAE-C) column. The neutral polysaccharide in the mixture is first eluted by an appropriate running buffer; the acidic polysaccharide is then eluted at a higher salt concentration.

Neutral polysaccharides later separated into α-glucans (adsorbed fraction) and β-glucans (nonadsorbed fraction) with the help of gel filtration and affinity chromatography. This process now allows for the highly specific and efficient purification of some carbohydrates. The complete purification process of G. lucidum polysaccharides is given in Figure 3.

Figure 3: Purification of polysaccharides by chromatography.

Conformational properties and analytical techniques

Polysaccharides having hyper-branched structures, to characterize such structures for their chemical structure and chain conformations are not an easy task. The chemical structures were analyzed by FTIR spectroscopy, Raman spectroscopy, NMR spectroscopy- Liquid-state NMR (1D and 2D) and Solid-state NMR, several chromatographic techniques like Gas Chromatography (GC), GC– Mass (GC–MS) and High-Performance Liquid Chromatography (HPLC) were employed for fractionation of polysaccharides. Chain conformations of polysaccharides in solutions were investigated using static and dynamic light scattering, viscosity analysis based on the theory of dilute polymer solutions, and Atomic Force Microscopy (AFM) including single molecular AFM and AFM-based single-molecule Force Spectroscopy, fluorescence correlation spectroscopy and NMR spectroscopy.

Characterization of Polysaccharides

The chemical structures of polysaccharides, such as the sugar composition, type of glycosyl linkage and the branched structures, were characterized by spectral analysis, chemical analysis and chromatography.

FTIR spectroscopy

FTIR spectroscopy technique was used in investigating the vibrations of molecules and polar bonds between the different atoms. Fourier transform infrared (FT-IR) spectroscopy is a physicochemical method based on measurement of vibration of a molecule excited by IR radiation at a specific wavelength range. Functional groups present in a molecule tend to absorb IR radiation in the same wavenumber range regardless of other structures in the molecule, and spectral peaks are derived from the absorption of bond vibrational energy changes in the IR region. Structures of polysaccharides, such as monosaccharide types, glucosidic bonds and functional groups, can be analyzed using FTIR spectroscopy [37,38]. In the range of 1100–1010 cm^-1, three strong absorption peaks appear for pyranoside, and two peaks for furanoside.

Raman spectroscopy

Compared with FTIR spectroscopy, Raman spectroscopy is highly sensitive to detect the vibrations of molecules and non-polar bonds of the same atom. Raman spectroscopy is the best suitable technique to characterize the helical conformation and the plane fold of bio-macromolecules [39]. The Raman spectra of saccharides segregated into four regions: the bands in the range of 350-600 cm^-1 are assigned to skeletal modes of pyranose rings; the anomeric region is from 600 to 950 cm^-1, the glycosidase stretching modes appear in the region 950–1200 cm^-1; and the CH₂ and C–OH deformations region is from 1200 to 1500 cm^-1.

NMR spectroscopy

NMR spectroscopy has become the most powerful and non-invasive physicochemical technique for determining polysaccharide structures providing detailed structural information of polysaccharides, including identification of monosaccharide composition, elucidation of α- or β-anomeric configurations, the establishment of linkage patterns, and sequences of the sugar units in polysaccharides.

Liquid-state NMR

The liquid-state NMR has become recognized as an important developing tool for chemical structural analysis of polysaccharides [40]. Most polysaccharides can be dissolved in water and dimethyl sulfoxide (DMSO), thus denatured water and DMSO (D₂O and DMSO-d6) are common solvents for polysaccharides in the liquid-state NMR experiments. The proton signals of polysaccharides overlap in the range of 3.5-5.5 ppm in the 1H NMR spectrum, it is difficult to assign them. Leeuwen et al, [41], investigated the ¹H NMR spectroscopy of the primary structural characterization of α-D-glucans in detail, in which chemical shift patterns for (α1→2)- , (α1→3)-, (α1→4)- and (α1→6)-linked D-glucose residues were analyzed. In contrast, the range of ¹³C chemical shifts of polysaccharides is much wider than that of ¹H chemical shift, which comes from 60 to 110 ppm.

Solid-state NMR

Solid-state NMR in contrast with liquid-state NMR the line widths become broader mainly due to the anisotropic character and dipolar interaction [42]. The anisotropic parts of the interactions from the molecules can be removed when the solid sample rotates at 54.7°. Magic-angle-spinning (MAS) is essential to achieve high-resolution ¹³C solid-state NMR spectra [43]. The intensity of the solid 13C signals can be enhanced using cross-polarization (CP) technology, in which the polarization transfers from ¹H to ¹³C. In recent years, solid-state NMR is used to analyze the chemical structures of polysaccharide to overcome the solubility problem, since the samples can be measured in a solid and dehydrated form. Spevacek and Brus, Pizzoferrato et al, [44,45], have reported the ratio between proteins and polysaccharides was directly determined through solid ¹³C CP/MAS spectroscopy.

Chromatography

The monosaccharide compositions, types of glycosidic linkages and branching of polysaccharides may be also analyzed by chromatography. GC, GC–MS and HPLC methods are employed after polysaccharides are hydrolysed by trifluoroacetic acid (TFA) or derived by the methylation, periodic acid oxidation and Smith degradation [46-49].

Chain Conformational analysis of Polysaccharides in Solution

Conformation of polysaccharides in solutions; especially in aqueous solutions, can be investigated according to the theory of dilute polymer solutions. The intrinsic viscosity η is a characteristic property of polysaccharide solution. Huggins and Kraemer's equations are used to estimate the η value by extrapolating to infinite dilution [50-52].

η_sp/C= η +K'η²C

(lnη_r)/C= η +K"η²C

Where K' is the Huggins constant and K" is the Kraemer constant, η_sp/C is the reduced specific viscosity, and (ln η_r)/C is the inherent viscosity.

Other Methods

The AFM-based single-molecule force spectroscopy (AFM–SMFS) technology is a powerful tool to characterize the force-induced conformational transitions, the dynamics, and supramolecular structures of polysaccharides at the molecular level [53-58].

Fluorescence correlation spectroscopy (FCS) is interesting to determine the conformations and sizes of polysaccharides at a lower concentration of about10^-8mol/l [59].

Biological Applications

Ganoderma lucidum has been used to treat various human diseases such as allergy, arthritis, bronchitis, gastric-ulcer, hyperglycemia, hypertension, chronic hepatitis, hepatopathy, insomnia, nephritis, neurasthenia, scleroderma, inflammation, and cancer. The fruiting bodies or spores of G. lucidum were linked to possible therapeutic effects (Table 2). The mechanisms of action involve the gut microbiota, meaning the polysaccharides act as prebiotics in the digestive system [60], Different compounds with various biological activities were extracted from mycelia. Current biological/biomedical applications of G. lucidum were given in Figure 4 [61-65].

Figure 4: Current biological/biomedical applications of G. lucidum.

 Table 2. Biologically active components in Ganoderma lucidum

Polysaccharides of G. lucidum have been used for a broad spectrum of health benefits from preventative measures and maintenance of health to the regulation or treatment of chronic as well as acute life-threatening illness. Nowadays more research is focussed on bioactive molecules from G. lucidum including polysaccharide as a chemotherapeutic agent to treat cancer [83]. In China, clinical trials on the approved drug are undergoing on G. lucidum polysaccharides to treat myopathy and other diseases [84]. Some of the significant biological/ biomedical applications of this mushroom were given in Table 3.

Table 3. Significant biological applications of Ganoderma lucidum polysaccharides

Conclusion

The Ganoderma lucidum mushroom is consumed commercially all over the world, because of its unique taste and curative properties. Because of the presence of numerous bioactive compounds, this mushroom is a popular herb as it contains a good amount of polysaccharides, which can be extracted by the ethanol-water solution. These polysaccharide molecules when absorbed into the human blood circulatory system, stimulates the immune modulators by activating the cellular and humoral components and increased production of macrophages. Based on the particle size and extraction time, the most common approach of hot water extraction (HWE) and ultrasound microwaveassisted extraction (UMAE), of water-soluble polysaccharides, is feasible economically. Also, the alkaline extraction of polysaccharides (AEP) of waterinsoluble polysaccharides resulted in good extraction yield as the alkaline treatment easily breaks down the dietary fibre of Reishi mushroom and speed up the release of polysaccharide extraction. The obtained polysaccharide extract is further purified by gel filtration and affinity chromatography technique that can be useful in scientific studies.

The fractionation of polysaccharides using new methodologies utilizing conformational properties was discussed that will open avenues for functional foods and herbal drugs. The biological preclinical studies showcasing the multiple health potentials of Ganoderma lucidum polysaccharides as antitumor, anti-inflammatory, antiviral, anticancer activities etc. were reviewed for future directions.

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