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) . 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,  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  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.
S. No.Method of Extraction% of YieldWater-soluble or in-solubleReference1HWE0.4SolubleChang and Lu [25
]2HWE7.5SolubleQian et al. [26
]3HWE3.7SolublePang et al. [27
by Supercritical CO2
2.98SolubleYu-Jie et al. [28
]5Breaking spores by fermentation using Lactobacillus plantarum
NANAChaiyavat, Chakkrapong and Sasithorn. [29
]6UMAE3.9SolubleSheng and Zheng [30
]7UAE2.07SolubleLiyan et al. [31
]8AEP8.21In-solubleSheng et al. [32
]9AEP1.41In-solubleJinghua et al. [33
]10AEP6.81In-solubleGao et al. [34
HWE: Hot Water Extraction; UMAE; Ultrasound Microwave Assisted Extraction; UAE: Ultrasonic Assisted Extraction; AEP: Alkaline Extraction of polysaccharides; NA: Not Available
Table 1. The percentage yield of polysaccharides extracted from Ganoderma lucidum using different
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,  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 .
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 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.
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 . 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 has become the most powerful and non-invasive physicochemical technique for determining polysaccharide structures providing detailed structural information of polysaccharides, including identiÃ¯Â¬Âcation of monosaccharide composition, elucidation of α- or β-anomeric conÃ¯Â¬Âgurations, the establishment of linkage patterns, and sequences of the sugar units in polysaccharides.
The liquid-state NMR has become recognized as an important developing tool for chemical structural analysis of polysaccharides . 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, , 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 in contrast with liquid-state NMR the line widths become broader mainly due to the anisotropic character and dipolar interaction . 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 . 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.
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].
ηsp/C= η +K'η2C
(lnηr)/C= η +K"η2C
Where K' is the Huggins constant and K" is the Kraemer constant, ηsp/C is the reduced specific viscosity, and (ln ηr)/C is the inherent viscosity.
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 .
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 , 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.
1(1→3)-β-D-glucansInhibition of growth of sarcoma S 180 tumour in miceSone et al. [66
(95% polysaccharides and 5% peptides)Activation of the immune response, stimulation of the IL-1β, IL-6, TNF-α, and IFN-γ production by macrophages and T lymphocytes, Inhibition of neutrophil apoptosis, Induction of neutrophil phagocytosis, Induction of GSTWang et al. [67
Hsu, Lee and Lin [68
Hsu et al. [69
Kim, Kacewand Lee [70
]3G009, amino polysaccharidesAntioxidantLee et al. [71
]4Glycoproteins (with fucose)Stimulation of IL-1, IL-2 and IFN-γ expression in spleen cellsWang et al. [72
]5GLIS, proteoglycansActivation of b-lymphocytesZhang et al. [73
]6CerebrosidesInhibition of DNA-polymeraseMizushina et al. [74
7Ganoderic acid (U, V, W, X, Y)Cytotoxic for hepatoma cellsShiao et al. [75
]8Ganoderic acid (A, C)Inhibition of farnesyl protein transferaseToth, Luu and Ourission [76
]9Lucidimol (A, B), Ganodermanondiol, Ganoderiol F, GanodermanontriolCytotoxic for sarcoma and lung carcinoma cellsMin et al. [77
El-Mekkawy et al. [78
Min et al. [79
]10Ganoderic acid FInhibition of angiogenesisKimura, Taniguchi and Baba [80
]11PhenolsAntioxidantMau, Lin and Chen [81
]12LipidsGrowth inhibition of hepatoma, sarcoma S-180 and reticulocyte sarcoma L-II in vivoLiu et al. [82
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 . In China, clinical trials on the approved drug are undergoing on G. lucidum polysaccharides to treat myopathy and other diseases . Some of the significant biological/ biomedical applications of this mushroom were given in Table 3.
S. No.Biological Applications of Polysaccharides from Ganoderma lucidum
Reference1Immunomodulation and potential antitumour activitiesZeng tao et al. [85
]2A novel polysaccharide from Se-enriched Ganoderma lucidum
induces apoptosis of human breast cancer cellsShang et al. [86
]3Effect of Reishi polysaccharides on human stem cells/progenitor cellsChen et al. [87
polysaccharides attenuate endotoxin-induced intercellular cell adhesion molecule-1 expression in cultured smooth muscle cells and the neointima in miceLin et al. [88
]5Ling-Zhi polysaccharides potentiate cytotoxic effects of anticancer drugs against drug-resistant urothelial carcinoma cellsHuang et al. [89
]6Immunomodulatory and adjuvant activities of a polysaccharide extract of Ganoderma lucidum
in vivo and in vitroLai et al. [90
]7The in vitro and in vivo experimental evidence disclose the chemopreventive effects of Ganoderma lucidum
on cancer invasion and metastasisChia and Gow [91
induced apoptosis in NB4 human leukaemia cellsEva et al. [92
]9The effects of Ganoderma alcohols isolated from Ganoderma lucidum
on the androgen receptor binding and the growth of LNCaP cellsJie, Kuniyoshi and Ryuichiro [93
]10Ganoderic acid T inhibits tumour invasion in vitro and in vivo through inhibition of MMP expressionNian, Jian and Jian [94
(Fr.) P. Karst enhances activities of heart mitochondrial enzymes and respiratory chain complexes in the aged ratSudheesh, Ajith and Janardhanan [95
]12An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and MAPK pathwaysLin et al. [96
]13Inhibitory effects of Ganoderma lucidum
on tumorigenesis and metastasis of human hepatoma cells in cells and animal modelsWeng et al. [97
induces the expression of CD40/CD86 on peripheral blood monocytesKazem and Majid [98
]15Effect of Ganoderma lucidum
on the activities of mitochondrial dehydrogenases and complex I and II of electron transport chain in the brain of aged rats.Ajith et al. [99
]16The signalling cascades of Ganoderma lucidum
extract in stimulating non-amyloidogenic protein secretion in human neuroblastoma SH-SY5Y cell linesPinweha et al. [100
]17Possible involvement of long-chain fatty acids in the spores of Ganoderma lucidum
(ReishiHoushi) to its antitumor activityFukuzawa et al. [101
]18Effect of Ganoderma lucidum
capsules on T lymphocyte subsets in football
Players on "living high-training low"Zhang et al. [102
]19Effects of Ganoderma lucidum
spores on HepG2 cells proliferation and growth cycleLi et al. [103
]20A randomized clinical trial of an ethanol extract of Ganoderma lucidum
in men with lower urinary tract symptomsNoguchi et al.[104
]21Serum amyloid A mediates the inhibitory effect of Ganoderma lucidum
polysaccharides on tumour cell adhesion to endothelial cellsYing et al. [105
]22The dual roles of Ganoderma antioxidants on urothelial cell DNA under carcinogenic attackYuen and Gohel [106
polysaccharides can induce human monocytic leukaemia cells into dendritic cells with immuno-stimulatory functionWing et al.[107
]24Effect of an extract of Ganodermalucidum
in men with lower urinary tract symptoms: a double-blind, placebo-controlled randomized and dose-ranging studyNoguchi et al.[108
]25Telomerase-associated apoptotic events by mushroom Ganoderma lucidum
on premalignant human urothelial cellsYuen, Goheland Au [109
polysaccharides in human monocytic leukaemia cells: from gene expression to network constructionKun et al.[110
]27Herbal mixtures containing the mushroom G. lucidum improve recovery time in patients with herpes genitalis and labialisHijikata, Yamada and Yasuhara [111
]28Androgen receptor-dependent and -independent mechanisms mediate Ganoderma lucidum
activities in LNCaP prostate cancer cellsZaidman et al. [112
polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expressionHua et al.[113
]30The potential of a novel polysaccharide preparation (GLPP) from Anhui-grown Ganoderma lucidum
in tumour treatment and immunostimulationPang et al.[114
polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblastHo et al.[115
]32Inhibition of oxidative stress-induced invasiveness of cancer cells by Ganoderma lucidum
is mediated through the suppression of interleukin-8 secretionThyagarajan et al. [116
]33Antitumor activity of extracts of Ganoderma lucidum
and their protective effects on damaged HL-7702 cells induced by radiotherapy and chemotherapyWang and Weng[117
]34Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1Lin et al. [118
]35Polysaccharide purified from Ganoderma lucidum
induces gene expression changes in human dendritic cells and promotes T helper 1 immune response in BALB/c miceYu et al. [119
extract inhibits proliferation of SW 480 human colorectal cancer cells.Xie et al. [120
extract stimulates glucose uptake in L6 rat skeletal muscle cellsJung et al. [121
]38Effects of water-soluble Ganoderma lucidum
polysaccharides on the immune functions of patients with advanced lung cancerGao et al. [122
Table 3. Significant biological applications of Ganoderma lucidum polysaccharides
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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