Chemical, biochemical, preclinical and clinical studies of Ganoderma lucidum polysaccharide as an approved drug for treating myopathy and other diseases in China

Abstract Ganoderma lucidum is an edible medicinal mushroom known as “Lingzhi” in China and “Reishi or Manetake” in Japan. It is a highly prized vitality‐enhancing herb for more than 2000 years. G. lucidum polysaccharide (GLPS) has been identified as one of the major bioactive components and developed into a drug named “Ji 731 Injection” in China since 1973. The large‐scale production of the drug began in 1985 and approved by the Chinese FDA as “Polysaccharidum of G. lucidum Karst Injection” (Ling Bao Duo Tang Zhu She Ye) in 2000, which is applied intramuscularly. After more than forty years of clinical use, its efficacy, safety and long‐term tolerability have been recognized by neurologists. It is one of a few non‐hormonal drugs used for treating refractory myopathy. It is also used for combination therapy, which reduces the amount of glucocorticoid required for myopathy patient who is in remission. In addition, it reduces adverse reactions and improves the quality of life for cancer patients during chemotherapy. We found 81 qualified chemical, biochemical, preclinical and clinical studies of GLPS both in English and in Chinese spanning from 1973 to 2017 by searching CNKI (China National Knowledge Infrastructure), Wanfang database and PubMed. The molecular mechanisms underlying GLPS's antioxidant, anti‐tumour, immune‐modulatory, hypoglycaemic, hypolipidaemic and other activities are discussed. Both preclinical and clinical studies are either deliberated or indexed in the current article. We aimed at providing a molecular picture as well as a clinical basis to comprehend GLPS as one of few polysaccharide‐based modern medicines with complicated chemical and pharmacological properties that prevent it from entering the world's market.

Ganoderma lucidum polysaccharide is the major component by weight among all constituents in the spores of G. lucidum. Over 200 polysaccharides have been isolated and structurally articulated in the fruiting bodies, mycelia and spores of G. lucidum; however, modern analytical chemistry is still revealing new polysaccharides from G. lucidum. 12 Glucose, mannose, galactose, xylose, fucose and arabinose have been identified in GLPS, and only b-glucan, a pure glucose polymer, is believed to be one of the active ingredients in GLPS. 10,16 The b-glucan structure in GLPS is shown in Figure 2.
Ganoderma lucidum is cultivated artificially on media of wood meal, rice bran and wood blocks in the last 10 years in China, Japan and the United States. However, these techniques do not guarantee a standardized yield as the compositions and structures of GLPS varied from batch to batch due to its non-template biosynthesis. Baskar et al 17,18 reported that using statistical and evolutionary optimization methods and response surface methodology could enhance the pro-  Table 1 shows the names, components, benefits, SFDA-certified numbers and the number of manufacturers that produce the specific G. lucidum-based drugs.
A G. lucidum polysaccharide-based product named "Ji 731 Injection" was used clinically for treating myopathy in China since 1973.  Polysaccharidum of G. lucidum Karst Injection is applied intramuscularly. At present, this product is used for treating neurosis, polymyositis, dermatomyositis, atrophic myotonia and muscular dystrophy, and various diseases caused by a defective immune system.
After more than forty years of clinical use, its efficacy, safety and long-term tolerability have been recognized by neurologists. It is one of the few non-hormonal drugs used for treating refractory myopathy. It is also used for combination therapy, which reduces the amount of glucocorticoid required for myopathy patient who is in remission. In addition, Polysaccharidum of G. lucidum Karst Injection reduces adverse reactions during chemotherapy and improves the quality of life of cancer patients significantly.
Based on CNKI (China National Knowledge Infrastructure), Wanfang database, and PubMed searches performed with keywords "Ganoderma lucidum polysaccharide," "Ji 731," "Ji Sheng," "Polysaccharide of G. lucidum Karst Injection" and "Ling Bao Duo Tang Zhu She Ye" both in English and in Chinese, we pulled out all of the publications. After reviewing the description about the GLPS used, the methods, data, results and conclusions, we found 81 qualified studies of GLPS both in English and in Chinese spanning from 1979 to 2017. A summary of the major biological activities of GLPS described in the qualified reports is shown in Figure 4, which demonstrates its immune-regulatory, antioxidant, anti-tumour, hypoglycaemic, hypolipidaemic, anti-myositis, anti-radiation, cardiac-protecting, sedative, hypnotic, lumbocrural pain relief, antidepressant, antibacterial, blood circulation-promoting, blood stasis-removing, hepatoprotective, anti-facial paralysis, anti-streptozotocin-induced diabetic nephropathy, anti-ageing, anti-bleomycin-induced pulmonary fibrosis and anti-chronic pancreatitis effects of GLPS.

| IMMUN OMODULATOR Y AN D ANTI-TUMOUR ACTIVITIES OF GLPS
Multiple studies demonstrated that GLPS is a potent immunomodulator that exerts a significant and comprehensive impact on immune cells including B lymphocytes, T lymphocytes, NKs, macrophages and Dendritic cells (DCs). These immunomodulatory effects are likely to have been mediated by its complex multiple components and can be one of the underlying anti-tumour mechanisms of GLPS to some F I G U R E 1 The picture of Ganoderma lucidum ZENG ET AL. GLPS from G. lucidum spores can increase the volume of macrophages and their ability to devour milk beads. 25 The pharmacological inhibitor assay showed that the ability of GLPS to enhance phagocytosis and chemotactic neutrophil function is mediated by PI3K, p38 MAPK, Src tyrosine kinase and Protein kinase C. 26 Chien et al 27 showed that the fucose-containing glycoprotein fraction (F3) isolated from the water-soluble extract of G. lucidum stimulated CD56+ NK activity in umbilical cord blood. After the F3 treatment, NK cell-mediated cytotoxicity is found to be significantly enhanced. The In general, all known polysaccharides have antioxidant activity that can be measured by mixing with a free radical compound DPPH. Several studies showed that GLPS can directly scavenge oxygen free radicals in a GLPS concentration-dependent manner. 29,30 Malondialdehyde (MDA) is a lipid peroxide formed when oxygen free radicals attack polyunsaturated fatty acids. Pretreatment of GLPS significantly reduced levels of ROS and MDA produced by myocardial cells; thus, GLPS plays a direct role in reducing myocardial oxidative stress. 31 Similarly, when PC12 cells are pre-treated for 2 hours with GLPS, it antagonizes the oxidative stress induced by Amyloid b  | 3281 serum and in hippocampus and protects rat neurons from oxidative stress. 33,34 GLPS also reduces lipid peroxidation in skeletal muscle of exhausted mice and ultimately protects the liver and skeletal muscle from oxidative stress. 16 In a rabbit model of liver ischemia-reperfusion injury, GLPS reduces the level of MDA significantly in liver and prevents liver from damage caused by oxygen free radical-mediated lipid peroxidation. 31 All animals generate multiple antioxidant enzymes, such as Glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD) and Catalase (CAT). GLPS not only increases the expression of GSH-Px, SOD and CAT that directly scavenges free radicals but also decreases the expression level of the enzymes that produce O À 2 , including NOX, XO and NOS. In fact, NOX is the main producer of ROS in vascular endothelial cells. [35][36][37] As shown in Table 2, GLPS increases the activities of skeletal muscle SOD and myocardial GSH-Px in mice and protects liver and skeletal muscle from damage in exhausted mice. 16 Moreover, GLPS enhances the activity of GSH-Px, SOD and CAT in serum and pancreas. 38,39 In an Alzheimer's disease model, GLPS stimulates the activity of SOD in serum and hippocampus in addition to accelerate the process of eliminating free radicals in the body. 35 GLPS also increases the serum level of SOD, GSH-Px and CAT in a mouse model of ovarian orthotopic tumour, indicating that GLPS enhances its sensitivity to chemotherapeutic drugs. 40 In the rabbit model of liver ischemia-reperfusion injury, GLPS significantly enhances the activity of SOD in liver of rabbit. 31 GLPS-treated rats have increased levels of serum and gastric tissue SOD, CAT and GSH-Px compared to that of control in a dose-dependent manner. 41 GLPS at >400 mg/kg significantly enhances the GSH-Px, CAT and SOD activities in the heart of type II diabetic rat and decreases myocardial MDA level at the same time. 42 GLPS increases SOD, GSH-Px and CAT levels in STZ-diabetic rats as well. 43 3.2 | Anti-tumour and immune-regulatory activities of GLPS Since 1970s, the anti-tumour effect has been demonstrated for GLPS. Joseph et al 44 found that GLPS at 100 mg/kg showed 80.8% inhibitory ratio of Ehrlich's ascites carcinoma (EAC) tumour cells (Table 3). Pang et al 45 found that GLPS at 100 and 300 mg/kg exhibits stronger growth inhibition against S 180 , Hepatoma solidity cell and EAC tumour cells. GLPS almost has no adverse reactions to human body, and this advantage is not possessed in many tumour chemotherapy drugs and other immune promoter. When combined with chemo-or radiation therapy, GLPS can improve the healthy state of cancer patients and strengthen anti-cancer effect of chemoor radiation therapy, which makes GLPS an excellent adjuvant therapeutic drug for cancer patients.

| Inhibiting tumour cell proliferation
Studies showed that GLPS does not kill cancer cells directly in vitro, [46][47][48] but it inhibits cancer cell proliferation, such as S 180 , PG and HL-60 cells, in vitro, when GLPS-treated animal serum is used in cell culture. It is proposed that one or more endogenous active substances are induced by GLPS, which are responsible for inhibiting cancer cell growth in vitro.

| Inhibiting tumour angiogenesis
Endothelial cell proliferation is one of the key steps in angiogenesis, which is essential for tumour growth. Thus, inhibition of vascular endothelial cell proliferation can inhibit tumour growth. As shown in Table 3

| Inhibiting tumour metastasis
Li et al and Liang et al 52,53 reported that GLPS has no cytotoxicity towards PC-3M in vitro, but GLPS could inhibit tumour cell adhesion and migration through the endothelium.

| Regulating the immune system
Studies showed that GLPS could enhance host immune function, activate the immune response and thereby inhibit tumour growth.
As shown in Table 3           Cardiac function and coronary flow were significantly better than mEC (P < .01) 72 mEC + Ji Sheng Injection The content of water and Malondialdehyde, in myocardium, was lower than mEC (P < .05) Activity of LDH and CK was lower than mEC (P < .01), and SOD was higher  Anti-skin ageing SD rats (10/group) Enhanced both hydroxyproline and SOD contents in a GLPS dose-dependent manner 83 Protective roles on bleomycin-induced pulmonary fibrosis SD rats Increased levels of glutathione, glutathione peroxidase, catalase and superoxide dismutase and decreased contents of malondialdehyde and hydroxyproline in the lung 90 Chronic pancreatitis ICR mice Alleviated the pancreatitis in mice through decreasing lipase, AMS, IFN-c and TNF-a levels as well as increasing SOD and total antioxidant activity 91 GLPS, Ganoderma lucidum polysaccharides; SOD, Superoxide dismutase.
| 3293 levels in STZ diabetic rats significantly and dose-dependently. 43 Huang et al 58,59 found that the high dose of GLPS is able to lower the levels of blood glucose, TC and TG and increase the level of HDL-C in diabetic rats. GLPS exerts noticeable hypoglycaemic and hypolipidaemic effects (P < .05) in diabetic rats, and all the beneficial effects are better than those of Cyclosporine A and N-acetyl-L-cysteamine (NAC). 60,61 Gong et al 62 also found that combined sporting with GLPS could lower the levels of blood glucose and lipids in diabetic mice.

MYOPATH Y
As shown in Table 5

STUDIES ON GLPS
As shown in Table 6, Polysaccharidum of G. lucidum Karst Injection has a curative effect on depression in a combination therapy. 70 When combined with glucocorticoids, Polysaccharidum of G. lucidum Karst Injection is effective in treating facial paralysis in children. 71 Zhang et al 72,73 conducted experimental studies on isolated heart preservation with Ji Sheng Injection and found that it can improve long-term preservation of the isolated arrested rat hearts. Acupoint injection of Ji Sheng Injection could treat lumbar hyperplasia and lumbocrural pain with improved efficacy. 74,75 When intravenously injected at 80 mg/kg to rats, GLPS could prolong sleeping time and improve the sleeping quality. 76,77 The level and activity of SOD is one of the important indicators to measure the body recover after irradiation. Studies demonstrated that GLPS could increase the level of SOD and the numbers of leucocytes. 78,79 Three independent studies have investigated the antibacterial activity of GLPS. [80][81][82] Lin observed the anti-skin ageing function of GLPS. In this model, GLPS increases both hydroxyproline and SOD contents in a concentration-dependent manner, indicating that GLPS slows the skin ageing process. 83 Furthermore, studies showed that GLPS is hepatoprotective 84 in that it corrects the metabolic abnormalities of diabetic mice and prevents or delays the progression of diabetic renal complications. 85 Finally, GLPS promotes blood circulation and removes blood stasis. 86 6 | FUTURE PERSPECTIVES GLPS has broader spectrum of therapeutic properties but lacks specific molecular targets. Interestingly, it was reported that among 656 US FDA-approved drugs tested, each drug hits more than 7 targets in the 73 total targets tested. 87 In addition, many drugs are less effective to the previously known targets compared to off-targets. Therefore, multiple or unknown biological targets in vivo might be a common but not a particular problem for GLPS.
In reality, multiple ingredients with multiple beneficial effects are essence of traditional Chinese medicines, which explains why GLPS is approved only by SFDA so far. However, there are multiple issues needed to be addressed before GLPS is accepted by governments and clinicians worldwide, such as how to comprehend the pharmacodynamics of GLPS, how to standardize the quality of GLPS and how to perform reliable pharmacokinetic studies of GLPS. Perhaps, the efficacy but not the homogeneity of polysaccharide-based drugs, such as GLPS, should be emphasized by the drug regulators worldwide in the near future.