Chinese FDA Approved Fungal Glycan-Based Drugs: An Overview of Structures, Mechanisms and Clinical Related Studies

Edible mushrooms have been used not only as food and nutraceuticals but also as important ingredients in traditional Chinese medicines for centuries. Pharmaceutical active components from different types of mushrooms have been extracted and studied by scientists all over the world during the past 50 years, and many biological functions, such as antitumor, immunomodulating, anti-oxidative, anti-inflammatory, and hypoglycemic activities, have been reported in peer reviewed English journals. Interestingly, the purified polysaccharides or glycans possess many reported functions of medicinal mushrooms, which make them potential drug candidates. However, glycans are a mixture of polysaccharides having variable numbers of monosaccharides, linkages, and molecular weight distributions as well as multiple biological functions that are hard to conceive as drugs by conventional standard in that a drug should have one structure and one function. On the other hand, multiple ingredients with multiple beneficial effects are essence of traditional Chinese medicines. Subsequently, glycans from different types of medicinal mushrooms are partially purified and trialed as oral and/or injectable drugs in China. Without serious safety concerns of mostly hot water extracted glycans from edible mushrooms and/or the cultured mycelium, eight of them are approved by Chinese Food and Drug Administration (SFDA) and used clinically in China since 1980s. This review article provides basic clinical information of the fungal glycan-based drugs in China and also summarizes structures, functions, and animal studies of fungal glycans conducted by scientists world-wide. Understanding glycan-based drugs at molecular biology level would be central for improving the clinical efficacy of current glycan-based drugs and for designing effective clinical trials of glycan-based drugs in future.


Introduction
Polysaccharides or glycans are located at intracellular, cell membrane, and extracellular spaces serving energy storage, structure, signal transduction, and system regulatory purposes in all living organisms. Among them, animal glycans have been extensively studied at genetic levels. Knocking out a series genes responsible for biosynthesis or modifications of glycans in different animal model systems reveals that animal glycans are indispensable for cell division [1], for animal development [2], and for maintenance of proper immunity and homeostasis in adult animals [3]. For example, endothelial heparan sulfate deficiency impairs L-selectin-and chemokine-mediated neutrophil trafficking during inflammatory responses [4]. Moreover, life-saving drug heparin, one type of glycans purified from animal tissues, remains to be an un-replaceable anticoagulant drug in modern medicine after 78 years of clinical use [2]. Furthermore, 20 different kinds of animal glycan-based drugs have preceded through clinical trials and are used clinically world-wide not only as anticoagulant but also used together with other conventional drugs for cancer treatment with an annual sale over $7 billion dollars [5].These facts indicate glycan-based drugs are not different from other biological drugs either from views of modern molecular biology or from views of their clinical importance.
Like animal cells, fungi synthesize several different types of glycans located in intracellular, cell wall, and extracellular spaces. Moreover, fungi possess several unique glycans that are not made by animal cells, such as chitin, β -glucans, and heteroglycans. In addition, glycanpeptide, glycan-lipid, and glycan-protein complexes isolated from fungi also have potent biological activities. This review article provides basic information of eight fungal glycan-based drugs in China and also summarizes peer-reviewed literatures about structures and biological functions of the fungal glycans at cell-and animal levels along with clinical studies that have been conducted by scientists world-wide.
As shown in Table 1, glycan contents of the eight approved drugs range from 30% to 93%. There are no specifications about monosaccharide compositions, glycan structures, molecular weight, or biological activity for these approved drugs due to inherent structural diversity of glycans. Taking Ganoderma lucidum glycans as an example, 16 different types of glycans with different monosaccharide compositions, different glycan linkages, and different molecular weight have been purified and identified by applying additional purification schemes when hot water extracted glycans are used as starting materials (Table 2). Therefore, these drugs are not "pure" or a single type of glycans. [8] [9] Ref. [ Β -Glucans are glycans that contain only glucose as structural components and are linked with β -glycosidic bonds. β -glucans are the simplest and the most studied fungal glycans. The biologically active fungal β -glucans are those comprising β (1,3) linked-glucose with side-chains of glucose with β (1,6) linkage. As shown in Table 2, the six β -glucans purified from Ganoderma lucidum are either water soluble or insoluble with molecular weight ranged from 5.2 x 10 3 to 1.0 x 10 5 Da. Therefore, β -glucans are not pure glycans. β -glucans can bind to six identified receptors on cell surface of immune cells ( Figure 1) [66-73]. The β -glucan and receptor interactions can activate multiple signaling transduction pathways directly or indirectly through macrophages, monocytes, dendritic cells, natural killer cells, B-cells, T-cells and neutrophils. β -Glucans also stimulate the release of cytokines, such as tumor necrosis factor (TNF-α) and several interleukins.
Activating the immune cells explain the immunomodulating and antitumor activities of β -glucans. However, not only fungi but also bacteria, plants, and algae synthesize biological active β -glucans. Moreover when present in animal blood circulation, tissues, or organs, most foreign glycans are recognized as sign of bacterium or fungus invasions through a series of receptors on immune cells including those receptors that bind to β -glucans. Most of glycans also have antiinflammatory properties that could not be explained by the glycan/ receptor signal transduction mechanism. Moreover, anti-oxidative and hypoglycemic properties are also common to all biological active glycans. As shown in Table 1, the drug indications of the 8 fungal glycan-based drugs are very different, which suggests the unique glycan compositions, but not the β -glucans alone, might contribute to the observed pharmaceutical effects of the fungal glycan-based drugs.
Being not pure might be a reasonable character for glycan-based drugs since glycans produced by nature are never pure even for the simplest β -glucans (see different characteristics of six β -glucans purified from Ganoderma lucidum listed in Table 2. We will briefly review the structure, function, clinical uses, and animal studies (summarized in Table 3) of the 8 fungal glycans and the glycan-based drugs in the next section.

Structural and Functional Studies of Fungal Glycans Lentinan from Lentinus edodes
Lentinan is a name given to β -glucans purified from Lentinus edodes. The antitumor property of lentinan was first reported by Chihara et al in 1970 [74]. Sasaki and Takasuka demonstrated that the primary structure of lentinan has β -(1-3)-glucose backbone with many (1-6)-β -glucose branches [ In 1985, lentinan is approved as an adjuvant for stomach cancer therapy in Japan. The lentinan activates immune cells [76], promotes the T-and B-lymphocyte proliferation, and enhances the activities of NK cells. The lentinan also plays multiple roles in inducing α-interferon production and leukocyte infiltration into tumor tissues [77]. The biological activities of lentinan have been studied by using mouse-,  rat-, chicken-, and pig-based animal models [76, 78-88]. These animal studies confirm that lentinan stimulate the productions of different cytokines and have antitumor and immunomodulating properties.

Polyporus glycan
Polyporus glycan is extracted from the sclerotium of Polyporus umbellatus. The major component of polyporus glycan is a β -glucan with a (1-3)-β -glucose backbone and (1-6)-β -glucose side chains with a molecular weight of approximately 1.  [92,93]. It is also used in treating leukemia and liver cancers [94,95]. Study has also shown that Polyporus glycans are effective in protecting liver from certain toxins [95]. Polyporus glycans are also used together with chemotherapy drugs to treat primary lung cancer, liver cancer, cervical cancer, nasopharyngeal carcinoma, esophageal cancer and leukemia.

Polysaccharide-K (PSK) or krestin
PSK or Krestin is a protein bound glycan isolated from cultured mycelium of Polystictus Versicolor. Glucose is the major monosaccharide found in PSK. PSK also contains arabinose, rhamnose, fucose, galactose, mannose, and xylose [96]. The glycans in PSK is highly branched. The molecular weight of PSK is around 1 × 10 5 Da and the protein component is covalently linked to the β-1,6 glucose side chain. PSK has increased the survival time of cancer patients in randomized, control studies, with stomach cancer (meta-analysis of 8,009 patients) [97], colorectal cancer (randomized, controlled study

Models Effects
Ref.

Mouse
Have radiation protection properties [154] Mouse Increase plasma insulin level and the activities of hepatic hexokinase and glucose-6-phosphatase dehydrogenase, and decrease hepatic glucose-6-phosphatase level [155] Rat Improve cognitive function via regulation of the CREB signaling pathway and cholinergic system in the hippocampus [156,157] Rat Increase cholinergic activity [158] Rat Increase fecal neutral steroids, total bile acids excretion, and SCFA productions [159]  of 448 patients) [98], but PSK has produced mixed results with liver cancer [99]. Rat-based animal studies confirmed the anti-metastasis properties of PSK [100][101][102].
Three mechanisms are proposed to explain the clinical effectiveness of PSK in suppressing cancer relapse [103]. First, PSK improves host immune-competence by inhibiting the production and also by neutralizing immunosuppressive substances that are increased in cancer. Second, PSK activates immune cells such as lymphocytes, either directly or by regulating the production of various cytokines. Third, PSK acts directly on cancer cells. In addition, the effects of PSK on the production of various cytokines and nitric oxide (NO) have also been reported [104,105].

Ganoderma Sinensis glycans
Ganoderma sinensis glycans are purified from fruiting body of Ganoderma Sinensis. The major bioactive Ganoderma sinensis glycans are α/β -glucans, glycan-protein complex and water-soluble heteroglycans with different combinations of glucose, mannose, galactose, xylose, fucose as well as arabinose. The molecular weight of the glycans ranges from 10 3 to 10 6 Da [106].
Ganoderma sinensis glycan-based drugs are available as capsules.
Published reports indicated the drug is used for neutralizing mushroom poisoning [10] and stimulate leukocytes productions in leukopenia patients [11] . Further studies showed that Ganoderma sinensis glycans enhance the immune responses in patients with advanced-stage cancer [107,108]. Ganoderma sinensis glycans also have potent antioxidant activities [109][110][111]. Mouse-based animal studies indicate that Ganoderma sinensis glycans enhance the levels of a variety of citokines [112].

Maitake glycans
Grifola Frondosa is also called maitake. A bioactive β -glucan fraction termed D-fraction was isolated from both mycelia and fruiting body of Grifola Frondosa by Japanes mycologists in 1984. Grifolanis of maitake glycans is a glycan-protein complex, which is called Wu Rong D-fraction in China. Its glycans mainly consist of glucose along with xylose, fucose, galactose, and mannose. The ratio of protein to glycan in Grifolanis is 7:3. The average molecular weight of Grifolanis is greater than 1x 10 6 Da. Only Wu Rong D-fraction have antitumor activities [145].
Grifola Frondosa glycans used for drug production is isolated from cultured mycelium. The glycan-based drugs are available as capsules. Published reports indicated the drug is used for cancer treatment [56,57], impaired glucose tolerance conditions [58], and for treating polycystic ovary syndrome [59]. Grifola Frondosa glycans have also been used for cosmetic and other biological purposes [146].

Future Perspectives
There are multiple issues needed to be addressed before fungal glycan-based drugs are accepted by governments and clinicians worldwide, such as how to comprehend the pharmacodynamics of fungal glycan-based drugs at molecular level, how to standardize quality, composition, purity of the highly dispersed glycan molecules, and how to perform reliable pharmacokinetic studies. Compared to conventional small molecule-and protein-based drugs, the advantages of glycan-based drugs are their broad spectrum of therapeutic properties, relatively low toxicity, less drug-resistant issues, and relatively low costs. The disadvantages of glycan-based drugs are the inherited heterogeneity of their structures and functions, lack of tools to do proper structure analyses, and difficulty in establishing structure and function relationships. Thus, developing reliable biological assays and novel structural characterization tools might be critical in understanding the information encoded in the fungal glycans and to perform reliable pharmacokinetic and pharmacodynamic studies.