Ganoderma: The mushroom of immortality

Ganoderma is the genus from order Aphyllophorales with more than 300 species. Ganoderma contains various compound that showed many biological activities e.g. enhancer of immune system, antitumor, antimicrobial, anti-inflammatory, antioxidant and acetyl cholinesterase inhibitory action. These bioactive compounds related to the triterpenoids and polysaccharides classes. Proteins, lipids, phenols, sterols, and others, are also recorded. Ganoderma is currently an important source in the pharmaceutical industry and is one of the most promissing projects in the world of food and medicine, which is being highlighted these days. In Egypt seven species of Ganoderma were recorded by Abdel-Azeem (2018). Among cultivated mushrooms, G. lucidum is unique in that its pharmaceutical rather than nutritional value is paramount. A variety of commercial G. lucidum products are available in various forms, such as powders, dietary supplements, and tea. These are produced from different parts of the mushroom, including mycelia, spores, and fruit body. The commercial cultivation of Ganoderma in Egypt not started yet. In this article the most important bioactive materials produced by Ganoderma, their applications and business opportunity in Egypt will be discussed.

basidiospore (Donk 1964). In all, 219 species within the family have been assigned to the genus Ganoderma, of which G. lucidum is the species type (Moncalvo 2000). Basidiocarps of this genus have a laccate (shiny) surface that is associated with the presence of thick-walled pilocystidia embedded in an extracellular melanin matrix (Moncalvo 2000). Ganoderma species are found all over the world, and different characteristics, such as shape and color (red, black, blue/green, white, yellow, and purple) of the fruit body, host specificity, and geographical origin, are used to identify individual members of the species (Zhao and Zhang 1994;Woo et al. 1999;Upton 2000).

Major bioactive components in Ganoderma
Ganoderma contains a wide variety of bioactive molecules, such as terpenoids (Fig. 2), steroids, phenols, nucleotides and their derivatives, glycoproteins, and polysaccharides. Mushroom proteins contain all the essential amino acids and are especially rich in lysine and leucine. The low total fat content and high proportion of polyunsaturated fatty acids relative to the total fatty acids of mushrooms are considered significant contributors to the health value of mushrooms (Chang and Buswell 1996;Borchers et al. 1999;Sanodiya et al. 2009).

Triterpenes
The Terpenes are a class of naturally occurring compounds whose carbon skeletons are composed of one or more isoprene C5 units. Terpenes have also been found to have antiinflammatory, antitumorigenic, and hypolipidemic activity. Terpenes in Ginkgo biloba, rosemary (Rosemarinus officinalis), and ginseng (Panax ginseng) are reported to contribute to the healthpromoting effects of these herbs (Mahato and Sen 1997;Mashour et al. 1998;Haralampidis et al. 2002). The triterpenes (Fig. 3) are a subclass of terpenes and have a skeleton of C30 and have molecular weights ranging from 400 to 600 kDa and their chemical structure is complex and highly oxidized (Mahato and Sen 1997;Zhou et al. 2007). In Ganoderma the chemical structure of the triterpenes is based on lanostane, which is a metabolite of lanosterol, the biosynthesis of which is based on cyclization of squalene (Haralampidis et al. 2002). Extraction of triterpenes is usually done by means of methanol, ethanol, acetone, chloroform, ether, or a mixture of these solvents. The extracts can be further purified by various separation methods, including normal and reverse-phase HPLC (Chen et al. 1999;Su et al. 2001).
The first triterpenes isolated is from G. lucidum called the ganoderic acids A and B, which were identified by Kubota et al. (1982). Since then, more than 100 triterpenes with known chemical compositions and molecular configurations have been reported to occur in G. lucidum. Among them, more than 50 were found to be new and unique to this fungus. The vast majority are ganoderic and lucidenic acids, but other triterpenes such as ganoderals, ganoderiols, and 49 ganodermic acids have also been identified (Nishitoba et al. 1984;Sato et al. 1986;Budavari 1989;Gonzalez et al. 1999;Ma et al. 2002;Akihisa et al. 2007;Zhou et al. 2007;Jiang et al. 2008;Chen et al. 2010).

Antitumor anticancer activity
The triterpene extracts of G. lucidum are known to induce apoptosis of multiple human cancer cell lines. However, the cytotoxic activity of triterpenes varied significantly across different subtypes of triterpenes. Most triterpenoids extracted and identified from Ganoderma have shown robust biological activities. The ganoderic acids isolated from Ganoderma have shown antiviral, anticancer, antioxidant, hepatoprotective, cytotoxic, antiplatelet aggregation, and inhibition of histamine release and hypocholesterolemic activities. The most abundant triterpenic acid from G. lucidum is GA T which shows significant anticancer activity both in vivo and in vitro experiments. GA has been found to inhibit tumor invasion by inhibiting matrix metalloproteinase (MMP)-9 expressions.
Another triterpenic acid GAD has been shown to directly bind to 14-3-3ζ protein and this binding may contribute to the facilitation of apoptosis observed in human epithelial cell line (HeLa) cell.The triterpene extracts identified from G. lucidum and other Ganoderma species have shown anticancer property under in vivo conditions. The carcinogenic effects shown by various types of extracts from G. lucidum include various cancer cell lines (breast, colon, lung, pancreas, prostate, and skin). The known mechanisms through which the extracts of G. lucidum exhibit anticancer activities include direct inhibition of cell proliferation through cancer-specific cell cycle arrest and apoptosis. Various other studies have also shown that the triterpene extracts of G. lucidum have antioxidant activity and have the potential to reduce oxidative damage by directly scavenging free radicals generated in the cell due to the increase in the activity of superoxide dismutase and catalase which are enzymes involved in removing harmful free radicals and ROS. Smina et al. (2011) revealed in mice that triterpenes from G. lucidum showed antioxidant activity which may be due to increased activity of antioxidant enzyme sand they further observed that total terpenes from G. lucidum prevent radiation-induced DNA damage and apoptosis in splenic lymphocytes of mice under in vitro conditions. In a recent study carried by by Smina et al. (2016), total triterpenes from G. lucidum were highly effective in reducing the levels of lipid peroxidation and protein oxidation to near normal values in both liver and brain tissues in Swiss albino mice under in vivo conditions. Total triterpenes, when administered under vivo conditions, were also found to be successful in restoring the antioxidant enzyme activities and glutathione level in liver and brain of irradiated mice. Administration of total triterpenes, before radiation exposure, significantly decreased the DNA strand breaks. The inhibitory activity of triterpenoids isolated from Ganoderma species against HIV has also been reported by Cassels and Asencio (2011). The ganoderic acid isolated from G. lucidum showed inhibitory effects on the replication of hepatitis B virus (HBV) in HepG2215 cells (HepG2-HBV-producing cell line) over 8 days. Production of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) were, respectively, 20% and 44% of controls without ganoderic acid treatment .

Anti-inflammatory potential
In the present study we evaluated the anti-inflammatory effects of the triterpene extract from G. lucidum (GLT) in LPS-stimulated macrophages. Here we show that GLT markedly suppressed the secretion of inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and inflammatory mediator nitric oxide (NO) and prostaglandin E2 (PGE2) from lipopolysaccharide (LPS)-stimulated murine RAW264.7 cells. GLT also down-regulated LPS-dependent expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW264.7 cells. The anti-inflammatory effects of GLT were mediated by the inhibition of transcription factor NF-κB as demonstrated by decreased NF-κB-DNA binding activity, and the suppression of p65 phosphorylation in LPS-stimulated macrophages treated with GLT. Moreover, GLT inhibited LPS-dependent AP-1-DNA binding activity and downregulated expression of AP-1 subunit c-Jun. In addition, GLT suppressed the activity of MAP kinases as observed by the down-regulation of LPS-induced phosphorylation of ERK1/2 and JNK but not p38. In vivo experiments clearly demonstrated that GLT also inhibited the production of TNF-α and IL-6 in LPS-induced endotoxemic mice. Apart from its antiinflammatory activity, GLT suppressed cell proliferation of RAW264.7 cells through cell cycle arrest at G0/G1-G2M, which was mediated by the down-regulation of expression of cell cycle regulatory proteins cyclin D1, CDK4 and cyclin B1, respectively. In conclusion, the antiinflammatory and anti-proliferative effects of GLT on macrophages are mediated through the inhibition of NF-κB and AP-1 signaling pathways. ( Dudhgaonkar s. et al 2009 )

Polysaccharides extracted from Ganoderma
Various polysaccharides have been extracted from the fruit body, spores, and mycelia of Ganoderma they are produced by fungal mycelia cultured in fermenters and can differ in their sugar and peptide compositions and molecular weight. G. lucidum polysaccharides (GL-PSs) are reported to exhibit a broad range of bioactivities, including anti-inflammatory, hypoglycemic, antiulcer, antitumorigenic, and immunostimulating effects (Miyazaki and Nishijima 1981;Hikino et al. 1985;Tomoda et al. 1986;Bao et al. 2001;Wachtel-Galor et al. 2004). Polysaccharides are normally obtained from the mushroom by extraction with hot water followed by precipitation with ethanol or methanol, but they can also be extracted with water and alkali. Structural analyses of GL-PSs indicate that glucose is their major sugar component (Bao et al. 2001;Wang et al. 2002). However, GL-PSs are heteropolymers and can also contain xylose, mannose, galactose, and fructose in different conformations, including 1-3, 1-4, and 1-6-linked β and α-D (or L)-substitutions (Lee et al. 1999;Bao et al. 2002).
Branching conformation and solubility characteristics are said to affect the antitumorigenic properties of these polysaccharides (Bao et al. 2001;Zhang et al. 2001). The mushroom also consists of a matrix of the polysaccharide chitin, which is largely indigestible by the human body and is partly responsible for the physical hardness of the mushroom (Upton 2000).
Numerous refined polysaccharide preparations extracted from G. lucidum are now marketed as over-the-counter treatment for chronic diseases, including cancer and liver disease .Various bioactive peptidoglycans have also been isolated from G. lucidum, including G. lucidum proteoglycan (GLPG; with antiviral activity; , G. lucidum immunomodulating substance (GLIS;Ji et al. 2007), PGY (a water-soluble glycopeptide fractionated and purified from aqueous extracts of G. lucidum fruit bodies; Wu and Wang 2009), GL-PS peptide (GL-PP; Ho et al. 2007), and F3 (a fructose-containing glycoprotein fraction; Chien et al. 2004).

Other beneficial components extracted from Ganoderma
Phosphorus, sulfur, potassium, calcium, and magnesium are their main mineral components. Iron, sodium, zinc, copper, manganese, and strontium were also detected in lower amounts, as were the heavy metals lead, cadmium, and mercury (Chen et al. 1998). Significantly, no cadmium or mercury was detected in these samples. G. lucidum can also contain up to 72 μg/g dry weight of selenium and can bio transform 20-30% of inorganic selenium present in the growth substrate into selenium-containing proteins (Du et al. 2008).
Germanium was fifth highest in terms of concentration (489 μg/g) among the minerals detected in G. lucidum fruit bodies collected from the wild (Chiu et al. 2000). This mineral is also present in the order of parts per billion in many plant-based foods, including ginseng, aloe, and garlic (Mino et al. 1980). Although germanium is not an essential element, at low doses, it has been credited with immunopotentiating, antitumor, antioxidant, and antimutagenic activities (Kolesnikova et al. 1997). G. lucidum contains some other compounds that may contribute to its reported medicinal effect, such as proteins and lectins.
The protein content of dried G. lucidum was found to be around 7-8%, which is lower than that of many other mushrooms (Chang and Buswell 1996;Mau et al. 2001). Bioactive proteins are reported to contribute to the medicinal properties of G. lucidum, including LZ-8, an immunosuppressive protein purified from the mycelia (Van Der Hem et al. 1995); a peptide preparation (GLP) exhibiting hepatoprotective and antioxidant activities (Sun et al 2004) and a 15-kDa antifungal protein, ganodermin, which is isolated from G. lucidum fruiting bodies (Wang and Ng 2006).
The carbohydrate and crude fiber content of the dried mushroom was examined and found to be 26-28% and 59%, respectively (Mau et al. 2001). Lectins were also isolated from the fruit body and mycelium of the mushroom (Kawagishi et al. 1997), including a novel 114-kDa hexameric lectin, which was revealed to be a glycoprotein having 9.3% neutral sugar and showing hem agglutinating activity on pronase-treated human erythrocytes (Thakur et al. 2007). Lectins (from the Latin word legere, which means to pick up, choose) are non-enzymatic proteins or glycoproteins that bind carbohydrates. Many species of animals, plants, and microorganisms produce lectins, and they exhibit a wide range of functions. In animals, for example, lectins are involved in a variety of cellular processes and the functioning of the immune system (Wang et al. 1998).
Also Metalloprotease, which delays clotting time; ergosterol (provitamin D2); nucleosides; and nucleotides (adenosine and guanosine) (Wasser et al. 2005;Paterson 2006). Kim and Nho (2004) also described the isolation and physicochemical properties of a highly specific and effective reversible inhibitor of α-glucosidase, SKG-3, from G. lucidum fruit bodies. Furthermore, G. lucidum spores were reported to contain a mixture of several longchain fatty acids that may contribute to the antitumor activity of the mushroom (Fukuzawa et al. 2008 ;Wachtel-Galor et al. 2011).

Economic importance of Ganoderma mushroom
Cultivation of the Genoderma mushroom in the countries of Asia is of great importance to its health benefits as well as the huge economic return from it. For a new business opportunity in Egypt, there are many factors which will help this industry to success, including the appropriate climate, spread of Ganoderma in the governorates of the Nile Delta region, the availability of suitable areas for cultivation (no need of special difficult preparations), the low cost and the most important factor are the studies and researchers who are able to develop this project and provide sufficient information to help the workers in it. The Ganoderma project can provide employment opportunities for young people, eldery and women for ease of operation, providing foreign currency where the kilogram sold for about 36 dollars, also opening a new market in the field of pharmaceutical industries. In addition, strengthening the market with a natural product has 52 enormous benefits as reishi coffee and tea both for prevention and treatment of diseases. For more details please read Mansour et al. (2018).

Conclusion
There is still little information available concerning the use of advanced fermentation techniques for obtaining Ganoderma lucidum mycelium and its valuable components in Egypt. A better understanding of its morphology and physiology is crucial for the development of large scale fermentation processes. Over the past two decades, the Ganoderma industry has developed greatly and today offers thousands of products to the markets. Despite the large market, there are problems with the industry which prevent it from establishing an effective market. In Egypt the world Ganoderma cultivation, products, industry and research is urgently needed.