Ganoderma

Ferreira, Isabel C. F. R., Heleno, Sandrina A., Reis, Filipa S., Stojkovic, Dejan, Queiroz, Maria João R. P., Vasconcelos, M. Helena & Sokovic, Marina, 2015, Chemical features of Ganoderma polysaccharides with antioxidant, antitumor and antimicrobial activities, Phytochemistry 114, pp. 38-55 : 45-51

3.1. Ganoderma polysaccharides

Among the Ganoderma genus, there are several reports in the literature describing the antioxidant activity of polysaccharides isolated from G. lucidum (Li et al., 2007; YouGuo et al., 2009; XiaoPing et al., 2009; Liu et al., 2010; Kao et al., 2011; Ma et al., 2013; Shi et al., 2013; Zhonghui et al., 2013) ( Table 1 ).

Homo-glucans and hetero-glucans isolated from this species have promising radical scavenging abilities, as evaluated by several in vitro antioxidant assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power, chelating ability, hydroxyl radical scavenging activity, 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) scavenging activity, superoxide radical scavenging activity and hydrogen peroxide scavenging activity, respectively ( Liu et al., 2010; Ma et al., 2013; Shi et al., 2013) ( Table 1 ). A low molecular weight β-1,3-glucan (LMG) was able to significantly increase the viability (from 40% to 80%) of a mouse leukaemic monocyte macrophage cell line (RAW 264.7) with H 2 O 2 -induced oxidative stress, reduced reactive oxygen species (ROS) formation and also suppressed the activities of neutral and acidic sphingomyelinases (SMases) (Kao et al., 2011). A homo-polysaccharide composed by mannose also had very interesting antioxidant activity under in vitro and in vivo conditions. This polysaccharide displayed promising free radicals (O 2 —; HO. and DPPH) scavenging ability and was able to increase the activity of the antioxidant enzymes, SOD (from 67.4 to 115.4 U/mL and 140 to 230 U/mL), CAT (from 7.82 to 13.91 U/mL and 13.0 to 22.0 U/mL) and GSH-Px (from 10.42 to 26.39 U/mL and 16.0 to 36.0 U/mL), as well as decrease malondialdehyde (MDA) levels (from 16.0 to 8.0 mmol/mL) in rats with cervical and ovarian carcinomas (YouGuo et al., 2009; XiaoPing et al., 2009). Zhonghui et al. (2013) studied the antioxidant capacity of a G. lucidum polysaccharide (GL-PS) against exercise-induced oxidative stress, which was related with the dose; the activity of the antioxidant enzymes significantly increased: SOD (from 110 to 170 U/mg protein), CAT (from 1.58 to 1.95 U/mg protein) and GSH-Px (from 6.0 to 15.0 U/ mg protein), while the levels of MDA decreased (from 8.2 to 4.8 nmol/mg protein). A hetero-glucan also isolated from G. lucidum showed antioxidant activity against mitochondria oxidative injury induced by γ- irradiation, causing a drastic decrease in MDA (from 1.24 to 0.55 nmol/mg protein), lipid hydroperoxides (LOOH) (from 1.09 to 0.04 nmol/mg protein) and protein carbonyl formation (from 0.84 to 0.22 nmol/mg protein), while protein thiol formation increased (from 9.28 to 13.42 nmol/mg protein). This hetero-glucan also increased the activity of the antioxidant enzymes SOD (from 3.07 to 6.11 U/mg protein), CAT (from 3.25 to 7.08 U/mg protein) and GSH-Px (from 2.66 to 4.77 U/mg protein) (Li et al., 2007). The main linkages in the homo-glucans were β-(1–3), (1–4) and (1–6) glycosidic bonds, as also in hetero-glucans, composed of different sugars, such as mannose, glucose, rhamnose, galactose, galactose, xylose, arabinose and fucose in different proportions. Liu et al. (2010) isolated a homo-glucan and a hetero-glucan, both low molecular weight polysaccharides, and reported a higher antioxidant activity of the homo-glucan because of its lower molecular weight. Nevertheless, Ma et al. (2013) isolated hetero-glucans with different molecular weights, and the polysaccharide with the highest molecular weight gave the highest antioxidant activity.

3.2. Ganoderma glycopeptides

There are several reports on the in vitro and in vivo antioxidant activity of glycopeptides obtained from Ganoderma species (Yu-Hong and Zhi-Bin, 2002; Zhang et al., 2003; Sun et al., 2004; Zhao et al., 2004; Chen et al., 2008; Jia et al., 2009; Li et al., 2009, 2010, 2011, 2012a).

The most abundant component isolated from G. atrum (PSG-1) is a glycoprotein with a molecular weight of 1.013 KDa, composed of 10.1% of protein with 17 general amino acids, and different sugars namely, mannose, galactose and glucose linked by O -glycosidic linkages (Chen et al., 2008). PSG-1 was studied for its antioxidant activity against anoxia/re-oxygenation injury in neonatal rat cardiomyocytes, anoxia/re-oxygenation-induced oxidative stress in mitochondrial pathway, oxidative stress induced by D - galactose in mouse brain, and age-related oxidative stress in mice. The authors reported very potent antioxidant activity by protecting cardiomyocytes from anoxia/re-oxygenation. It significantly increased the activity of antioxidant enzymes, decreased the levels of MDA, and attenuated ROS formation, thereby having the potential to promote health and improve aging-associated pathologies by modifying the redox system and improving the immune function (Li et al., 2009, 2010, 2011, 2012a).

Yu-Hong and Zhi-Bin (2002) studied the antioxidant activity of a glycopeptide (GLP) isolated from G. lucidum against the injury of macrophages induced by ROS. It was composed of 14 amino acids, D- rhamnose, D- xylose, D- fructose, D- galactose, D- mannose, and D- glucose as sugars, linked by β- glycosidic linkages, and with a molecular weight of 0.585 KDa. GLP showed in vitro and in vivo antioxidant activity by increasing the survival rate of macrophages, and protecting the mitochondria against injury by membranepermeant oxidant (t BOOH). GLP was also studied for its antioxidant activity on streptozotocin (STZ)-diabetic rats, being able to increase non-enzymatic and enzymatic antioxidants, serum insulin level and to reduce lipid peroxidation (Jia et al., 2009).

Sun et al. (2004) studied GLP antioxidant activity in different oxidation systems (soybean and lard oils as oxidation substrates), and described an excellent activity comparable to the synthetic antioxidant butylated hydroxytoluene (BHT) in soybean oil. This glycopeptide was able to block soybean lipoxygenase activity, showed scavenging activity toward hydroxyl radicals produced in a deoxyribose system, quenched superoxide radical ion produced by pyrogallol autoxidation, displayed antioxidant activity in rat liver tissue homogenates and mitochondrial membrane peroxidation systems, and also blocked the auto-hemolysis of rat red blood cells.

A glycopeptide isolated from G. lucidum, with a molecular weight of 0.5849 KDa, composed of 17 amino acids and rhamnose, xylose, fructose, galactose, mannose and glucose as sugars, linked by β- glycosidic linkages, had antioxidant activity by reducing ROS formation, MDA levels and increasing the activity of manganese superoxide dismutase in rat cerebral cortical neuronal cultures exposed to hypoxia/re-oxygenation (Zhao et al., 2004). This glycopeptide also showed antioxidant activity (free radicals scavenging ability) by protecting against alloxan-induced pancreatic islets damage under in vitro and in vivo conditions (Zhang et al., 2003).

3.3. Crude polysaccharidic Ganoderma extracts

The antioxidant activity of crude polysaccharidic extracts obtained from Ganoderma specie, have been recently described (Shi et al., 2010; Yang et al., 2010; Heleno et al., 2012; Kozarski et al., 2012; Zhao et al., 2012; Pan et al., 2013).

A polysaccharidic extract from G. lucidum showed antioxidant activity in rats with gastric cancer by increasing the activity of antioxidant enzymes (SOD, CAT and GSH-Px) (Pan et al., 2013). Other polysaccharidic extracts, also obtained from G. lucidum, displayed radicals scavenging ability, reducing power and lipid peroxidation inhibition, with the extract obtained from spores as the most effective (Heleno et al., 2012). Kozarski et al. (2012) reported the antioxidant activity of polysaccharidic extracts from G. applanatum and G. lucidum namely, radicals scavenging activity, reducing power, lipid peroxidation inhibition and chelating abilities.

Zhao et al. (2012) reported the radio-protective effects of a G. lucidum polysaccharidic extract on mouse deoxyribonucleic acid (DNA) damage induced by cobalt-60 gamma-irradiation, and described that DNA strand-break and micronuclei frequency were significantly reduced, while GSH-Px activity and nucleated cell count in bone marrow significantly increased. This polysaccharidic extract also increased SOD activity and decreased MDA levels.

Polysaccharidic extracts prepared from G. lucidum also lowered serum levels of MDA and intercellular adhesion molecule- 1 in heart and liver of mice with ischemic reperfusion, and increased antioxidant enzymes activity (Shi et al., 2010). In diabetic rats, the polysaccharidic extract was able to reduce oxidative injury and inhibit apoptosis by increasing antioxidant enzymes activity, and modifying B-cell lymphoma 2 (bcl-2) expression and bcl-2- associated X protein (bax)/bcl- 2 ratio (Yang et al., 2010).

The studies performed over the last decades concerning antioxidant properties of polysaccharides, glycoproteins and crude extracts described that the radicals scavenging activity seems to be mostly related with the increase in the activity of antioxidant enzymes such as SOD, CAT and GSH-Px (Yu-Hong and Zhi-Bin, 2002; XiaoPing et al., 2009; YouGuo et al., 2009; Pan et al., 2013).

There are not many studies on the antioxidant activity of Ganoderma polysaccharides, and the existing ones only report polysaccharides from G. lucidum. Most of these studies were carried out under in vitro conditions; and reports using in vivo assays are scarce and do not describe the mechanism of action involved. Instead, they only describe an increase in antioxidant enzymes activity after exposure to a specific injury. Additionally, those polysaccharides were isolated but not completely chemically characterized. The available data generally include molecular weights and, in some cases, sugars composition; glycosidic linkages are rarely characterized. Therefore, it is not possible to highlight a key chemical feature directly related with the antioxidant activity of Ganoderma polysaccharides, since there is a lack of information on their chemical characteristics. Based on the existing reports with available information about structural features, it can only be speculated that homo-glucans and hetero-glucans with β (1 + 3) glycosidic linkages have strong antioxidant properties (Liu et al., 2010; Kao et al., 2011).

4. Antitumor Ganoderma polysaccharides

4.1. Ganoderma polysaccharides

The crude water-soluble extract of G. lucidum has been used in traditional Chinese medicine as antitumor and immunomodulating agent (Zong et al., 2012). Most reports concerning the antitumor activity of polysaccharides from Ganoderma demonstrate that it is mainly related to the host-mediated immune function (Gao et al., 2005a; Paterson, 2006). Ganoderma polysaccharides have received special attention from the scientific community, especially those from the species G. lucidum , and their antitumor activity has been studied both in vitro and in vivo.

Hence, bioactive polysaccharides have been isolated from the fruiting bodies of G. lucidum (Bao et al., 2002; Zhao et al., 2010) and from the mycelia cultivated in liquid culture medium (Kim et al., 1993; Peng et al., 2005; Liu et al., 2012). Some polysaccharides have also been isolated from the culture medium of growing mycelium (extracellular polysaccharides) (Sone et al., 1985).

Antitumor effects of polysaccharides isolated from G. lucidum , such as the branched heteroglucan, arabinoxyloglucan (GL-1), were initially observed in subcutaneously transplanted sarcoma-180 ascites growing in mice (Miyazaki and Nishijima, 1981; Table 2 ). This polysaccharide contains a backbone and side-chains involving D- glucopyranosyl, α-(1 + 4), β-(1 + 6) and β-(1 + 3) linkages; arabinose is present as a part of the non-reducing terminal residues, and xylose is present as a part of the side-chain. This hetero-glucan strongly inhibited the growth of sarcoma-180 solidtype tumor (inhibition ratio, 95.6–98.5%) after intra-peritoneal injection (20 mg /kg) for 10 days in imprinting control regions (ICR) of mice (Miyazaki and Nishijima, 1981; Table 2 ). Sone et al. (1985) also described the antitumor activity of G. lucidum polysaccharides either from the fruiting bodies or the mycelium against sarcoma-180 solid tumor. Once again, the studied polysaccharides had (1 + 3)-β- D-glucan bonds and some (1 + 4)-linked glucosyl units ( Table 2 ).

The antitumor potential of Ganoderma polysaccharides is usually related to their immunomodulatory activity. Since polysaccharides have a large molecular weight, these compounds cannot penetrate cells, but they bind to immune cell receptors. It has been proven that there are fungal pattern-recognition molecules for the innate immune system. However, the mechanism by which the innate immune system recognizes and responds to fungal cell wall carbohydrates is a very complex and multifactorial process (Lowe et al., 2001).

Yan and collaborators suggested that the activity of polysaccharides from G. lucidum was mediated through the complement receptor type 3 (CR3 receptor), which binds β- glucan polysaccharides (Yan et al., 1999) . Indeed, G. lucidum polysaccharide (GLP), known as a homo-glucan from G. lucidum , isolated by hot aqueous extraction and ethanol precipitation from the fruiting bodies of this medicinal mushroom, exerted its antitumor activity in sarcoma-180 solid tumor by inducing a cascade of immuno-modulatory cytokines. It could induce a marked increase in the gene expression levels of IL-lα (2-fold), IL-lβ (3-fold), TNF-α (2-fold), IL-12 p35 (up to 6-fold), and IL-12 p 40 in the splenocytes. In the macrophages, GLP promoted a remarkable increase in the gene expression levels of IL-lβ (2.5- to 3-fold), TNF-α (up to 6-fold), and granulocyte–macrophage colony-stimulating factor (GM-CSF) (up to 2-fold) (Ooi et al., 2002; Table 2 ). GLP also exhibited antitumor effects on solid tumor induced by Ehrlich’s ascites carcinoma cells. Indeed, 100 mg /kg of this polysaccharide showed 80.8% and 77.6% reduction in tumor volume and tumor mass, respectively, when administered 24 h after tumor cell implantation. Moreover, GLP with the same dose but administered prior to tumor inoculation, showed 79.5% and 81.2% inhibition of tumor volume and tumor mass, respectively (Soniamol et al., 2011). GLP not only has (1 + 3)-β- D- glucan bonds, but also has (1 + 6)-β- D branches. Furthermore, structural features such as (1 + 3)-β- linkages in the main chain of the glucan, and additional (1 + 6)-β- branch points, seem to be important factors for the observed antitumor activity.

The same features were verified for the heteroglucans from G. tsugae described by Peng et al. (2005), which were composed by (1 + 3)-β- D- glucans and (1 + 4)-α- D- glucans and also possess antitumor activity against sarcoma-180 solid tumor ( Table 2 ). Actually, the fruiting body of G. tsugae is used to promote health and longevity in Oriental countries (Haghi, 2011), which can be, in part, justified for these findings.

More recently, other heteropolysaccharides from Ganoderma have been studied both in vivo and in vitro, establishing inhibitory activity in tumor cell lines, apoptosis induction and inhibition of tumors transplanted in mice (Liu et al., 2012; Zhang et al., 2012; Ma et al., 2013; Table 2 ).

Other polysaccharides from G. lucidum with immunomodulatory properties have been described, namely PG-1 and PG-2, which increased the proliferation and pinocytic activity of macrophages and played an inhibitory effect on the growth of a human breast cancer cell line (MDA-MB-231) (Zhao et al., 2010; Table 2 ).

There are also reports on the antitumor potential of other polysaccharides from Ganoderma species but without their chemical characterization.For example, other authors reported the antitumor properties of mannogalactoglucans and (1 + 3)-β- glucuronoglucans from G. lucidum tested in vitro (in cell lines) and of glucogalactans from G. tsugae tested in vivo (pre-clinical animal models), through their immunomodulatory activity (Zhuang et al., 1994; Cho et al., 1999; Wasser, 2002; Moradali et al., 2007; Zhang et al., 2007; Ferreira et al., 2010).

4.2. Ganoderma polysaccharide-protein or -peptide complexes

As mentioned above, polysaccharides isolated from Ganoderma may be also bound to protein or peptide residues. These polysaccharide-protein or -peptide complexes have also been described as having antitumor properties. G. lucidum polysaccharide peptide (GLPP), potently inhibited human lung carcinoma cell line (PG), proliferation in vitro and reduced the xenograft (of the PG cell line) in albino laboratory-bred strain of the house mouse (BALB/c) nude mice in vivo. This compound proved to have anti-angiogenic activity, which can be the basis of its antitumor effects. This polysaccharide-peptide with relative molecular weight (MW) of 512500, is composed by D- rhamnose, D- xylose, D- fructose, D- galactose, and D- glucose linked together by β- glycosidic linkages (Cao and Lin, 2004).

A fucose-containing glycoprotein fraction from the water-soluble extract of G. lucidum seems also to be responsible for its immunomodulating and antitumor activities through the stimulation of the expression of cytokines, especially IL-1, IL-2 and IFN-γ (Wang et al., 2002). Although the active fraction contained the majority of D- glucose, D- mannose and D- galactose, the only active component identified in the glycopeptide fraction contained fucose residues. In addition, the crude extract of G. lucidum did not stimulate expression of cytokines, whereas the glycoprotein fraction significantly induced expression of IL-1, IL-2, and IFN-γ (Wang et al., 2002).

A well-known proteoglycan from G. lucidum is the previously mentioned GLIS (Section 2.1). This proteoglycan with a molecular weight of about 2000 kDa, and carbohydrate portion consisting of hetero-polysaccharides composed predominantly of D- glucose, Dgalactose and D- mannose, exhibits an effective antitumor effect by increasing both humoral and cellular immune activities (Zhang et al., 2010).

A water-soluble protein-bound polysaccharide from the fruiting bodies of G. atrum (PSG-1), besides the antioxidant properties previous reported, displayed potent antitumor activity in sarcoma180 transplanted mice by induction of tumor apoptosis through mitochondrial pathways, and its antitumor effect was related to immuno-enhancement (Li et al., 2011a). This compound, proved to improve immunity by inhibiting proliferation of a mouse colon carcinoma cell line (CT26) via activation of peritoneal macrophages. In vivo, PSG-1 considerably suppressed the tumor growth in CT26 tumor-bearing mice (Zhang et al., 2013).

A G. lucidum polysaccharide-peptide conjugate with a molecular weight of 0.5125 KDa and polysaccharide chain assembled in β- glycosidic linkages, also exhibited antitumor potential in different studies. For example, it significantly inhibited tumor growth in a murine sarcoma180 model, and inhibited proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) by inducing cell apoptosis and decreasing the expression of secreted vascular endothelium growth factor (VEGF) in human lung cancer cells (Li et al., 2008; Cao and Lin, 2006).

4.3. Ganoderma polysaccharidic extracts /fractions

Polysaccharidic fractions from Ganoderma have also been described as having potential antitumor activity. Ganopoly is one of the most well-known aqueous polysaccharidic fractions from G. lucidum with antitumor potential. Treatment of mice with Ganopoly for 10 days could significantly reduce tumor weight in a dose-dependent manner in S-180-bearing mice. Furthermore, the polysaccharide caused significant cytotoxicity in the human tumor cell lines: Human Caucasian Cervical Epidermoid Carcinoma (CaSki), Human Cervical Cancer (SiHa), Human Hepatoma (Hep3B), Human Hepatocellular Liver Carcinoma (HepG2), Human Colon Carcinoma (HCT116) and Human Colon Adenocarcinoma Grade II (HT29) Cells in vitro, with marked apoptotic effects observed in CaSki, HepG2 and HCT116 cells (Gao et al., 2005a). Other studies showed that Ganopoly could enhance immune responses in patients with advanced-stage cancer, which could be an approach for overcoming immunosuppressive effects of chemotherapy/ radiotherapy (Gao et al., 2003b, 2005b).

Some studies also suggest that antitumor activity of polysaccharides from fresh fruiting bodies of G. lucidum (PS-G), is achieved through stimulation of the production of IL-1β, TNF-α, and IL-6 from human monocyte-macrophages and IFN-γ from T lymphocytes. These studies were carried out in the human promyelocytic leukaemia (HL-60), and human lymphoma cell lines (U937) (Wang et al., 1997). G. lucidum polysaccharide (GL-B), consisting of seven fractions of polysaccharides isolated from this species, was tested both in vitro (HL-60, and sarcoma-180 cells), and in vivo (sarcoma-180 cells injected sub-dermally into the axillary fossa of the right foreleg of BALB/c mice), and this established that its antitumor potential is also related to TNF-α and IFN-γ (Zhang and Lin, 1999). Co-administration of G. lucidum polysaccharides and cyclophosphamide potentiated the antitumor activity of this drug (used to treat cancer and immune diseases) in mice. These results indicate that either G. lucidum or its active components have antitumor activity in mice, and that Ganoderma polysaccharides have a synergic effect on the antitumor activity of cyclophosphamide (Lin and Zhang, 1999).

G. tsugae mycelium and fruiting body polysaccharidic fractions have also been investigated. Seven glycans with strong antitumor activities were obtained from 14 water-soluble and 15 water-insoluble fractions extracted from G. tsugae fruiting bodies. The bioactivity against sarcoma-180/mice was tested, and tumor inhibition ratios from 26.1% to 100% were observed (Wang et al., 1993). Water-soluble fractions were protein-containing glucogalactans associated mainly with mannose and fucose, but also containing arabinose and rhamnose; water-insoluble fractions represented protein-containing β-(1 + 3)-glucans with different protein content and some of them with (1 + 6)-β- D- glucosyl branched chains. The molecular weight averages ranged from 8 × 10 3 to 700 × 10 3 (Wang et al., 1993). Sixteen water-soluble polysaccharides were extracted from G. tsugae mycelium and examined for their antitumor effects on sarcoma- 180 in mice (Zhang et al., 1994). The active polysaccharides obtained were: (i) a glycan-protein complex containing 9.3% protein, with a hetero-glyco-chain of mannose and xylose; (ii) a glucan-protein complex containing 25.8% protein and (iii) a glycan-protein with glucose as the main component, and associated with arabinose, mannose, xylose, and galactose. The molecular weight ranged from 10 × 10 — 3 to 16 × 10 — 3 (Zhang et al., 1994). Comparison of active water-soluble polysaccharides obtained from the fruiting body and mycelium showed that the first were gluco-galactan-protein complexes, but those of the mycelium were homo-glucan-protein complexes or a hetero-glycan composed of mannose and xylose (Wasser, 2002). However, and once again, the structure with β-(1 + 3)-glucans and, in some cases, with (1 + 6)-β- D- glucosyl branched chains was present in these bioactive polysaccharidic fractions.

Other polysaccharidic fractions were also obtained from the water soluble extracts of G. applanatum . These preparations had antitumor properties against transplanted sarcoma- 180 in mice, and, for one of the obtained fractions, a complete regression of tumors was observed in more than half of animals; inhibition ratios were over 95%, with no sign of toxicity (Sazaki et al., 1971). These fractions were considered to be a glucan consisting partially of a mixture of β-(1 + 3) and (1 + 4) linked D- glucose residues.

Polysaccharidic extracts from the mycelium of G. lucidum also exhibited antitumor effects against fibrosarcoma in male and female mice and inhibited the metastasis of a lung tumor.

Different studies showed that bioactive polysaccharides and extracts could stimulate blood mononuclear cells to increase cytokines, tumor necrosis factor, interferon and interleukins production, induce apoptosis and meaningfully increased the lifespan of the tumor-implanted mice (Paterson, 2006; Ramberg et al., 2010; Roupas et al., 2012; Liao et al., 2013).

4.4. Structure-bioactivity relationship

Polysaccharides are one of the biologically active groups of compounds found in mushrooms, namely in Ganoderma genus, which have antitumor properties (Wasser, 2002; Lindequist et al., 2005; Paterson, 2006; Ferreira et al., 2010; Patel and Goyal, 2012; Nie et al., 2013). Thus, Ganoderma has been considered a bioactive therapeutic fungus (Paterson, 2006) and its antitumor potential has been explored (Wang et al., 1997; Yuen and Gohel, 2005).

The study and description of the chemical features of Ganoderma polysaccharides are very important as they allow us to infer or deduce structure-bioactivity relationships. Different polysaccharides from the Ganoderma genus have been isolated and characterized especially in the past three decades.

The first reports of Ganoderma polysaccharides structure date back to 1981, when Miyazaki and Nishijima characterized a water-soluble branched arabinoxyloglucan from G. lucidum , which contained β-(1 + 4)-, β-(1 + 6)- and β-(1 + 3)-D- glucopyranosyl residues in the backbone and side-chains. These authors inferred that the essential structure for the antitumor activity of polysaccharides from Ganoderma might be a branched glucan core involving (1 + 3)- β-, (1 + 4)-β- and (1 + 6)–β- linkages.

More recently, Bao et al. (2002) isolated three polysaccharides, two heteroglucans (PL-1 and PL-4) and one glucan (PL-3) from the fruiting bodies of the same species. This study showed that PL-1 had a backbone consisting of 1,4-linked α- D- glucopyranosyl residues and 1,6-linked β- D- galactopyranosyl residues with branches at O -6 of glucose residues and O -2 of galactose residues, composed of terminal glucose, 1,6-linked glucosyl residues and terminal rhamnose, respectively. PL-3 was a highly branched glucan composed of 1,3-linked β- D- glucopyranosyl residues substituted at O - 6 with 1,6-linked glucosyl residues. PL-4 was comprised of 1,3-, 1,4-, 1,6-linked β- D- glucopyranosyl residues and 1,6-linked β- D- mannopyranosyl residues. More recently, Wang et al. (2011) isolated five water-soluble heteropolysaccharides from the cultured fruiting body of G. lucidum , designated as GL-I to GL-V. These compounds proved to be heteropolysaccharides, mainly composed of glucose, galactose, mannose and arabinose. GL-I was the most branched of the heteropolysaccharides (27.0% degree of branching), while GL-V was mostly a linear glucan.

The biological activity/antitumor potential of polysaccharides seems to be highly correlated with their chemical composition and configuration, as well as their physical properties, being exhibited by a wide range of glycans extending from homopolymers to highly complex heteropolymers (Ooi and Liu, 1999). As stated initially by Miyazaki and Nishijima (1981), more recent studies continue to point to the importance of structural features such as (1 + 3)-β- linkages in the main chain of the glucan and additional (1 + 6)-β- branch points as essential factors for the antitumor activity of polysaccharides (Wasser, 2002) . Therefore, β- glucans containing mainly 1 + 6 linkages exhibit less activity, possibly due to their inherent flexibility of having too many possible conformations (Zhang et al., 2007; Ferreira et al., 2010). However, antitumor polysaccharides may have other chemical structures, such as hetero-β- glucans (Mizuno et al., 1995b), heteroglycan (Gao et al., 1996), β- glucan-protein (Kawagishi et al., 1990), α- manno-β- glucan (Mizuno et al., 1995b), α- glucan-protein (Mizuno et al., 1995b) and heteroglycan-protein complexes (Zhuang et al., 1993; Mizuno et al., 1996). It has been described that the antitumor activity of mushroom polysaccharides containing glucose and mannose may be due to their immunomodulating activity, since a polysaccharide receptor, which has been demonstrated to have high specificity for glucose and mannose, has been found on human macrophages (Lombard, 1994). Triple helical conformation of (1 + 3)-β- glucans is considered an important structural feature for their immuno-stimulating activity, but how the triple helical conformation of (1 + 3)-β- glucan precisely affects their antitumor action still remains unclear. Indeed, (1 + 3)-β- glucans exhibit antitumor activity related to their triple helical conformation (Zhang et al., 2007).

Higher antitumor potential seems to be also correlated with higher molecular weight (Mizuno et al., 1996; Wasser, 2002), lower level of branching and greater water solubility of β- glucans (Ferreira et al., 2010). Thus, although other features such as molecular weight or level of branching are very important for the antitumor potential of the polysaccharides, the molecules described above have the main glycosidic bonds required for this activity, which seems to be highly related with the results obtained.

The antitumor potential is the most explored bioactivity of Ganoderma polysaccharides , being extensively studied; the chemical structures are completely characterized, and even some mechanisms of action are proposed by some authors (Yan et al., 1999; Ooi et al., 2002). Analysing the available data, it can be highlighted that the essential structure for the antitumor potential of polysaccharides is a branched glucan core involving (1 + 3)-β-, (1 + 4)-β- and (1 + 6)-β- linkages (Miyazaki and Nishijima, 1981). Nevertheless, clinical human trails are needed to better understand the bioactivity of these interesting and extremely potent molecules, so that the investigation can progress in order to use these molecules in the development of new nutraceuticals or drugs.

4.5. Antioxidant and antitumor potential

The antitumor activity of Ganoderma , namely G. lucidum , seems to be also strongly related with its antioxidant properties, since water soluble polysaccharides extracted from G. lucidum were effective in preventing DNA strand breaks (Paterson, 2006).

An aminopolysaccharide fraction from G. lucidum (G009) was found to have the ability to protect against ROS, which is implicated in the pathophysiology of cancer (Pincemail, 1995). G009 inhibited iron-induced lipid peroxidation and inactivated hydroxyl radicals and superoxide anions. Furthermore, G009 also reduced oxidative DNA damage, suggesting that the aminopolysaccharide fraction of G. lucidum possesses chemopreventive potential (Lee et al., 2001).

Two cerebrosides (glycosphingolipids consisting of D- glucose, sphingosine and 2-hydroxypalmitoyl or 2-hydroxystearoyl fatty acid moiety, respectively), were also isolated from the fruiting body of G. lucidum (Mizushina et al., 1998) . Both molecules inhibited DNA polymerases, suggesting their possible use for cancer therapy by inhibiting DNA replication (Sliva, 2003).

With all the studies conducted so far, polysaccharides have been suggested to have an ability to enhance the host’s defense system in both antioxidant and antitumor abilities (Mizuno et al., 1995a; Pan et al., 2013). The work performed, especially in G. lucidum , indicates that fractions of polysaccharides were not as effective as their equivalent dose in the crude extract of the whole mushroom, suggesting that the bioactivity of this medicinal mushroom may be due to the synergistic effects of multiple compounds, such as triterpenes (Liu et al., 2002). This idea is supported by some studies, such as the study in which a polysaccharidic mixture containing isoflavone aglycones produced from the cultured mycelia of G. lucidum inhibited angiogenesis in BALB /c mice with implanted chambers containing a suspension of colon-26 cells (Miura et al., 2002).

5. Antimicrobial Ganoderma polysaccharides

5.1. Medicinal mushrooms as antimicrobial agents

Fungi are well known for the production of important antibiotic compounds, such as penicillin. However, the occurrence of antibiotics in the class of fungi known as mushrooms is less well documented (Miles and Chang, 1997). Mushrooms belong to the kingdom of Fungi, they were thought to have weak antifungal activities (Mizuno et al., 1995a,b) and therefore have rarely been investigated for their bioactivity as antifungal agents. It is only recently that they have become of interest due to their secondary metabolites exhibiting a wide range of antimicrobial activities.

Ganoderma species have been widely investigated for their therapeutic properties as antitumor and antiviral agents but have been far less investigated as a source of new antibacterial agents. A review by Gao et al. (2003a) on the antibacterial and antiviral value of Ganoderma species supported this observation, as there were few citations on research in this area. It is interesting to note that the majority of antibacterial investigations on Ganoderma species have been performed on the fruiting body and there are relatively few on extracts from the liquid cultivated mycelium.

5.2. Current antimicrobial research on Ganoderma species

Western and Eastern medicine have adopted different regulatory systems for herbal and mushroom preparations (Wasser, 2011). Western medicine has made little use of medicinal mushroom products partly due to their complex structure and lack of acceptable pharmacological purity (Sullivan et al., 2006). In the search for microbiologically active compounds from Ganoderma species, the majority of research has been performed on extracts from the fruiting body and mycelium, and there are a few studies on antimicrobial activity of isolated fractions or pure polysaccharides. It appears that there are a number of biologically active compounds to be found in the mycelium and fruiting body, but antimicrobial activity evaluation of chemically characterized polysaccharides is very limited. It could be only noted that (1 + 3)-β- D- glucan with (1 + 6)-β- D branches could act as antimicrobial agent in vivo.

5.3. Antibacterial activity of Ganoderma polysaccharides

The antibacterial activity of polysaccharides from G. lucidum fruiting bodies was reported ( Table 3 ) (Skalicka-Woźniak et al., 2012). Thirty six samples were analyzed. Four strains of G. lucidum (GL01, GL02, GL03 and GL04) were cultivated on the growth substrates of three different sawdust types: birch (Bo), maple (Kl) or alder (Ol) amended with wheat bran in three different concentrations: 10, 20 and 30% (w/w). Even though the richest in polysaccharides was the GL01 strain, the highest yields of the polysaccharides were observed in the GL04Kl3 sample (112.82 mg /g of dry weight). The antibacterial activity of the polysaccharides was determined in vitro using the micro-dilution broth method. A panel of eight reference bacterial strains was used and all the tested polysaccharides showed moderate antibacterial activity. The Micrococcus luteus American Type Culture Collection (ATCC) 10240 strain was the most sensitive with minimal inhibitory concentrations (MICs) 0.63–1.25 mg /mL. Nevertheless, the analyzed polysaccharides exhibited inhibitory effects against all the bacterial strains tested, with MICs ranging from 0.62 to 5.0 mg/mL. The minimal bactericidal concentrations (MBCs) of the samples were comparable (2.5 or 5.0 mg/mL). Only slight differences were observed between MICs and MBCs of the polysaccharide samples obtained from the strains of the G. lucidum fruiting bodies, and the ones obtained from the sawdust cultivation substrates. The low MBC/MIC ratios suggest that polysaccharides acted as bactericidal agents. The screening of antibacterial activity indicates that there were no significant differences in the antimicrobial activity between the polysaccharides obtained from the four strains of G. lucidum fruiting bodies and the ones obtained from different sawdust cultivation substrates. The polysaccharides tested exerted the strongest inhibitory effect towards M. luteus (MIC 0.62 or 1.25 mg /mL) (Skalicka-Woźniak et al., 2012).

In another study, G. lucidum polysaccharides were extracted with boiling water, and further tested for antimicrobial activity against three plant pathogens ( Erwinia carotovora , Penicillium digitatum , Botrytis cinerea ) and five food harmful microorganisms ( Bacillus cereus , Bacillus subtilis , Escherichia coli , Aspergillus niger and Rhizopus nigricans ) by the agar diffusion method. The results showed that the polysaccharide liquid had a powerful inhibitory effect on E. carotovora , a weak inhibitory effect on P. digitatum and a nearly non-inhibitory effect on B. cinerea , for the plant pathogens. Regarding food harmful microorganisms, the polysaccharide liquid had a strong inhibitory effect on B. subtilis and B. cereus , a weak inhibitory effect on E. coli and A. niger , and a nearly non-inhibitory effect on R. nigricans (Bai et al., 2008) .

Polysaccharides from the mycelia and basidiocarp of Ganoderma applanatum were found to possess activity against Acitenobacter aerogenes, Acrobacter aerogenes, Arthrobacter citreus, Bacillus brevis , B. subtilis, Corynebacterium insidiosum, E. coli , Proteus vulgaris , Clostridium pasteurianum , Micrococcus roseus , Mycobacterium phlei , Sarcina lutea and Staphylococcus aureus (Bhattacharyya et al., 2006) .

The extracellular polysaccharides obtained from Ganoderma formosanum culture medium were separated into three major fractions, PS-F1, PS-F2, and PS-F3, based on their molecular size (Wang et al., 2011a). Although the different monosaccharide’s composition in each fraction, D- mannose was the major constituent among all fractions, and in the two major fractions PS-F2 and PS-F3, the second most abundant sugar was D- galactose, followed by D- glucose. G. formosanum thus synthesizes a different form of polysaccharide as compared with other Ganoderma species (e.g., G. lucidum ) in which D- glucose is usually the major component (Wang et al., 2002). These results show that D- mannose and D- galactose are the major constituents of G. formosanum polysaccharides. The differences in carbohydrate composition among fungal polysaccharides could be due to strain variations or caused by different ways of cultivation (solid-state culture versus liquid-state culture). The polysaccharides were produced in a submerged mycelial culture. The fungal cell wall polysaccharides synthesized under different growth conditions may exhibit different biological effects. Methods of extraction may also affect the polysaccharides obtained from G. lucidum fruiting bodies, which could contain either β-1,3-glucans or α-1,4-linked polymannose (Miyazaki and Nishijima, 1981). It appears that both the sugar components and structures of the hetero-polysaccharides in the fungal cell wall are diverse and complicated. It is suggested that the extracellular polysaccharides of G. formosanum (PS-F2, and perhaps PS-F1 and PS-F3) have the potential to be used as immuno-stimulatory and antibacterial agents against Listeria monocytogenes injected in mice.

In the study of antibacterial activity of exopolysaccharide (EPS) from basal medium and malt medium obtained from different mushrooms, G. lucidum EPS showed the highest activity against the growth of B. cereus among other bacterial species (23 ± 0.61 mm and 18 ± 0.38 mm, respectively) (Mahendran et al., 2013).

Ganoderma polysaccharides have not been much studied regarding antimicrobial properties. Nevertheless, the available studies report mainly their activity against several pathogenic bacteria. Several authors reported antimicrobial activity of G. lucidum different extracts but not isolated polysaccharides (Sheena et al., 2003; Quereshi et al., 2010). Heleno et al. (2013) reported strong antibacterial, antifungal and also demelanizing properties of G. lucidum extract, even better than the standards ampicillin and streptomycin in some cases. Thus, polysaccharides isolated from these species should also be analyzed since they can have a strong participation in the antimicrobial properties exhibited by G. lucidum .

6. Ganoderma polysaccharides with non-reported bioactivity

Polysaccharides from Ganoderma species with previously non-reported bioactivity are briefly discussed here. The reported polysaccharides with non-tested bioactivity belong to alkali-soluble polysaccharides and/or water insoluble polysaccharides and water soluble polysaccharides ( Table 4 ).

The methods for isolation, purification and identification are given in Table 4 , as well as sugars composition and molecular weight. A water insoluble, but alkali-soluble glucan G-A was previously isolated from G. japonicum . (Ukai et al., 1982). Chen et al. (1998) have isolated water-insoluble glucans, namely GL4-1 and GL4-2 from the fruiting bodies of G. lucidum . A water soluble and low branched polysaccharide (SGL-III) was isolated from the spores of G. lucidum (Zhao et al., 2005) . A neutral, water soluble, heteropolysaccharide (GLPS3) was isolated from germinating spores of G. lucidum (Zhang et al., 2006) . A water soluble β- glucan (DESSK5) was reported in the basidiocarp of G. resinaceum (Amaral et al., 2008) . A water soluble polysaccharide, heteropolysaccharide LZ-C-1 was isolated from G. lucidum (Ye et al., 2009) . Neutral polysaccharide, soluble in water was isolated by Huang et al. (2011) from G. lucidum fruiting body. Novel heteropolysaccharides (GL-1 to GL-5) were also isolated from the fruiting bodies of G. lucidum (Wang et al., 2011a) . Finally, a novel water soluble and neutral β- D-glucan (GLSA50-1B) was isolated from the spores of G. lucidum (Dong et al., 2012) .

The chemical features of the described polysaccharides with non-reported bioactivity are similar to the ones described for the polysaccharides with some reported bioactivities. These substances might be further used for evaluation of its biological potential, as their chemical properties are promising.

Tables

Table 4 Polysaccharides isolated from Ganoderma species without reported bioactivity.

Ganoderma species	Origin	Extraction/isolation procedure	Identification	Polysaccharide type	Main glycosidic bonds	Sugars composition	Molecular	References
			technique				weight
G. japonicum (fruiting	Japan	Hot dichloromethane and hot	GLC-MS; 1H-	Alkali-soluble glucan	β-(1 + 3)-linked D-glucopyranosyl	Glucose	82000	Ukai et al.
body)	(wild)	methanol	NMR; IR; PC		residues with side-chains of single, β-	laminarabiose		(1982)
		Hot water; Dialysis; gel filtration			(1 + 6)-linked D-glucopyranosyl
		on Sepharose CL-4B			groups
G. lucidum (fruiting	China	PBS; Ethanol precipitations; 1 N	13C-NMR;1H-	Water insoluble glucans	(1 + 3)-α- D-glucans	Glucose	GL4–1 –	Chen et al.
body)	(cultivated)	NaOH	NMR; IR				1.95 × 10 5	(1998)
							GL4–2 –
							1.33 × 10 — 4
G. lucidum (spores)	na	Hot water followed by ethanol	13C-NMR; GC;	Water soluble	(1 + 3)-linked-Glc	Glucose	1.41 × 10 4	Zhao et al.
	(cultivated)	precipitation	GC–MS; IR	polysaccharide	(1 + 6)-linked-Gal	galactose		(2005)
					(1 + 4)-linked-Gal
					(1 + 6)-linked-Glc
G. tlucidum	na	Deproteinization by Sevag	GC; HPLC; IR;	Heteropolysaccharide	Na	Glucose	1.41 × 10 5	Zhang et al.
(germinating	(cultivated)	method and frozen-thaw	NMR			galactose		(2006)
spores)		method, fractionated by
		ultrafiltration and gel
		chromatography on CL-6B
		column.
G. resinaceum	Brazil	Chloroform–methanol; Hot	GC–MS; GPC;	Water soluble glucan	(1 + 3)-linked β- glucan	Glucose	2.6 × 10 4	Amaral et al.
(fruiting body)	(wild)	water; dialysis; Freeze–thawing;	NMR			mannose		(2008)
		ultrafiltration				galactose
						xylose
G. lucidum (fruiting	China	Hot water followed by ethanol	FT-IR	Water soluble	1,6-disubstituted-α-	Fucose	7000 Da	Ye et al.
body)	(wild)	precipitation; ultrafiltration	HPAPC	polysaccharide	galactopyranosyl, 1,2,6-	glucose		(2009)
			NMR		trisubstituted-α- galactopyranosyl,	galactose
					1,3-disubstituted-β- glucopyranosyl
					and 1,4,6-trisubstituted-β –
					glucopyranosyl residues
G. lucidum (fruiting	China	Utrasonic/microwave assisted	FT-IR	Water soluble neutral	Backbone: 1,4-disubstituted-β-	Glucose	2.5 × 10 6 kDa	Huang et al.
body)	(cultivated)	extraction	GC–MS	polysaccharide	glucoseopyranose and 1,4,6-	galactose		(2011)
			HPSEC		trisubstituted-β –glucoseopyranosyl
			NMR		Branched chains: 1,6-disubstituted-
					β- glucopyranosyl and 1,4-
					disubstituted-β–galactoseopyranosyl
G. lucidum (fruiting	China	Ethyl-acetate; Sevag	FT-IR	Water soluble	(1 + 4)-galactan heteropolysaccharide	glucose, galactose,	GL-I –	Wang et al.
body)	(cultivated)	method;Dialysis	GC–MS	Heteropolysaccharides	(1 + 3)-glucan;1,4,6-glucan, (1 + 3)-	mannose, arabinose	6.1 × 10 4	(2011)
			NMR		galactan, (1 + 6)-galactan, (1 + 4)-		GL-V –
					grabinan		10.3 × 10 4
					(1 + 3)-mannan, and/or (1 + 4)-xylan
					linkages
G. lucidum (spores)	China	Hot-water extraction, graded	GPL	Water soluble glucan	Backbone: 1,6-linked β- D-Glcp	Glucose	103 kDa	Dong et al.
	(cultivated)	ethanol precipitation, anion-	HPGPC		Side chain: 1,4-linked			(2012)
		exchange chromatography	NMR		Glcp residues

FT-IR – Fourier transform infrared spectrophotometer; GC – Gas chromatography; GC–MS – Gas chromatography coupled to mass spectrometry; GLC-MS – Gas liquid chromatography coupled

to

mass

spectrometry

;

GPC

–

Gel

permeation chromatography; HPGPC – High performance gel permeation chromatography; HPLC – High performance liquid chromatography; HPSEC – High pressure size exclusion chromatography

;

IR

–

Infrared

spectroscopy

;

NMR – Nuclear magnetic resonance spectroscopy; PC – Paper chromatography; na –data not available.