1. Antitumor Effects

Previous studies over the past 30 years have strongly suggested that there are three possible ways by which maitake (Grifola frondosa) exerts its anticancer effect—protection of healthy cells, prevention of tumor metastasis, and inhibition of tumor growth. In other words, maitake can fight against tumors directly and indirectly by enhancing the immune system. This section will mainly focus on the direct antitumor function of maitake, whereas the immunomodulatory effect will be discussed in the next section.

The antitumor activity of maitake was first reported by scientists in 1982, followed by their further study on the chemical structure of glucans extracted from the maitake fruiting body and their antitumor activity against Sarcoma 180 tumors in the Institute of Cancer Research (ICR). Scientists obtained different polysaccharide fractions and reported the D-fraction for the first time in 1988. Unlike many other antitumor polysaccharides derived from Basidiomycetes, which may become ineffective if administered orally, the D-fraction exhibited promising prospects because it could be administered orally, intravenously, and intraperitoneally. A nonrandomized clinical study was conducted to assess the effects of the D-fraction from maitake on 165 advanced cancer patients who received the D-fraction as crude powder tablets alone or in addition to chemotherapy. Results showed that maitake was effective against breast, liver, and lung cancers but less effective against leukemia, stomach, and bone cancers.

Further studies demonstrated that the D-fraction could function on mammary tumor cells directly through the modulation of different cellular processes during cancer development. A combination of the D-fraction (0.2 mg/mL) and vitamin C (0.3 mmol/L) was found to result in a 70% reduction in the viability of human hepatocarcinoma SMMC-7721 cells. Further purification of the D-fraction yielded the MD-fraction showed even better results than the D-fraction in terms of the inhibitory effect on mouse tumor growth. In addition to intraperitoneal injection in the test, the MD-fraction has also been demonstrated to inhibit tumor growth in mice via oral administration. Both the D-fraction and the MD-fraction were proven safe, with low or no toxicity.

Other polysaccharide fractions have also exhibited anti-tumor activity besides the D and MD fractions. The polysaccharide GFP-A, isolated from maitake, inhibited the proliferation of human colon cancer HT-29 cells in vitro, with 180 μg/mL as the most effective concentration. The polysaccharide fraction GFP-4, extracted from maitake, showed an inhibitory effect on human lung cancer cells at four °C. Due to structural changes, the inhibitory effect became lower after heat treatment at over 30 °C. The polysaccharides in maitake could regulate gene expression involved in the apoptosis of breast cancer cells so that cell proliferation was inhibited and the cell cycle was blocked.

In addition to polysaccharide fractions, the ergosterol derivatives from non-polar extracts of maitake were also found to have anti-proliferative effects on human tumor cells. Moreover, the ο-orsellinaldehyde component of submerged cultures of maitake exhibited tumoricidal activity against Hep 3B cells via apoptosis. Some glycoproteins isolated from maitakes, such as GFL and GFG-3a, also showed antitumor effects due to their anti-proliferative activity against cancer cells.

  1. Immunomodulation

Immunomodulation is the most well-known effect of maitake components and has been confirmed by many studies. These immunomodulatory components have been shown to enhance the actions of macrophages and many other immune-related cells, such as cytotoxic T-cells and natural killer (NK) cells. Furthermore, maitake components could increase the secretion of cytokines, which are signaling molecules, including interferons (IFN), interleukins (IL), tumor necrosis factors (TNF), and lymphokines with antiproliferative activity, causing apoptosis and differentiation in tumor cells, thus further increasing the efficiency of immune-related cells.

Polysaccharides have been recognized as the major immunomodulating components of maitake. The D-fraction is also a major polysaccharide fraction of maitake with significant immunomodulatory activity. The D-fraction could activate NK cells by upregulating their expression of TNF-α and interferon-gamma (IFN-γ) proteins. Meanwhile, the D-fraction also increased macrophage-derived IL-12, which further activated NK cells, implying that the D-fraction could provide long-term tumor-suppressive effects. The application of the D-fraction could reduce the effective dosage of the chemotherapeutic agent mitomycin-C (MMC) by increasing the proliferation, differentiation, and activation of immunocompetent cells. It could also reduce the immunosuppressive activity caused by MMC.

Apart from the D-fraction, other polysaccharide fractions with immunomodulatory activity have also been isolated from maitake. Insoluble and high-molecular-weight soluble forms of Grifolan (GRN) from maitake can activate macrophages by triggering cytokine secretion to produce TNF. Similarly, Mao et al. observed increased levels of TNF-α, IL-2, IL-1β, and nitric oxide (NO) in the serum with the dosage of polysaccharide GP11 from maitake, suggesting the activation of macrophages and the stimulation of tumoricidal activity. The anti-cancer activity of the polysaccharide fraction MZF from maitake was associated with the activation of cell-mediated immunity resulting from the induction of macrophage proliferation, increasing levels of IL12, IL2, IFN-γ, and TNF-α, as well as enhancement of NK cells and cytotoxic T lymphocytes. The GFP fraction promoted the production of cytokines and chemokines such as IL-6, IFN-γ, and TNF-α, and also effectively enhanced the proliferative activity of fibroblasts, contributing to immune-stimulating solid activity.

  1. Antiviral and Antibacterial Effects

There have been many studies reporting the beneficial effects of maitake in the treatment of viral infections, including those caused by hepatitis B virus (HBV), enterovirus 71 (EV71), and herpes simplex virus type 1 (HSV-1), and human immunodeficiency virus (HIV). A study on patients with chronic hepatitis B. showed that patients who took maitake fruiting body polysaccharides showed positive signs and a higher recovery rate than the control group. The MD-fraction from maitake could fight against HIV through several pathways, including direct inhibition of HIV, stimulation of the natural defense system against HIV, and reducing vulnerability to opportunistic infections. The GFP1 fraction was found to fight against EV71, the causative pathogen of hand-foot-and-mouth disease. The researchers found that maitake could hinder EV71 viral replication, suppressing genomic RNA synthesis and protein expression, and thus could be used as a promising therapeutic compound for EV71 treatment. In addition to polysaccharide fractions, the protein fraction GFAHP purified from maitake has also shown anti-viral effects. It significantly inhibited HSV-1 replication in vitro and reduced HSV-1-induced symptoms such as blepharitis in a murine model.

In addition to the antiviral effect, the D-fraction isolated from maitake has also shown antibacterial effects. The mechanism of antibacterial action of D-fraction was related to the immune-stimulating activity. The D-fraction could activate immuno-competent cells and induce the production of cytokines, which further lead to the activity enhancement of splenic T cells to kill Listeria monocytogenes. Unlike the antibacterial mechanism, the antiviral action of the D-fraction is not directly related to the immune system. The D-fraction interfered with HBV replication through the inhibition of HBV polymerase.

  1. Antidiabetic Activity

Multiple animal studies have demonstrated the hypoglycemic effects of maitake extracts. To test the antidiabetic activity of active ingredients in maitake, in vivo fasting serum glucose (FSG) or fasting blood glucose (FBG) measurements are generally performed after feeding the bioactive ingredients to animal models for 2 to 4 weeks. A high FSG level is one of the characteristics of diabetes mellitus sufferers. The influence on FSG level could directly indicate the antidiabetic effect of active ingredients in maitake.

The hypoglycemic mechanisms of these polysaccharide fractions are most likely to be linked to insulin activity. For instance, F2 and F3 polysaccharides and SX glycoprotein fractions have been suggested to exert a hypoglycemic effect through the insulin signal pathway. The SX-fraction could facilitate glucose uptake, activating the insulin receptor (IR) and insulin receptor substrate 1 (IRS-1), eventually resulting in increased insulin secretion. In a normal situation, a high glucose level would lead to low glucose uptake, but the SX-fraction overcame this suppressive effect and impaired the insulin signaling pathway. The hypoglycemic mechanisms of F2 and F3 polysaccharides were also related to IR and IRS-1. They could improve insulin sensitivity and decrease FSG levels by increasing protein levels of IR and IRS-1. The anti-diabetic effects of MT-α-glucan, such as ameliorating insulin resistance of peripheral target tissue and improving insulin sensitivity, were also reported to be associated with IR.

In addition to the enhancement of insulin activity, the hypoglycemic effects of maitake may be generated through the inhibition of α-glucosidase activity because an anti-α-glucosidase effect could prevent starch hydrolysis into disaccharides and decrease the blood glucose level. The hyperglycemic effects of non-polar fractions in maitake were examed both in vivo and in vitro. Research findings showed that maitake exhibited strong anti-α-glucosidase activity in vitro and could significantly lower the blood glucose level in high-fat-diet-fed and streptozotocin-induced hyperglycemic animals. The anti-α-glucosidase effect was attributed to the pyrrole alkaloids and ergosterols extracted from maitake, The ergosterol peroxide isolated from maitake contributed to its anti-diabetic effect.

  1. Lipid Metabolism Regulation and Anti-Hypertension Effects

The effects of maitake on lipid metabolism regulation and anti-hypertension have been shown in many reports. The maitake fruiting body as the feed, the triglyceride, cholesterol, and phospholipid levels in the serum of rats was suppressed by 30–80% compared with those of the control group of animals. Meanwhile, the weight of the extirpated liver was also 60–70% lower than that of the control group. The corresponding cholesterol excretion ratio in feces increased by 1.8 times with maitake treatment, further demonstrating that maitake treatment helped improve lipid metabolism and inhibit increases in liver lipid and serum lipid after the ingestion of high-fat feed. Similar results showed that serum total cholesterol concentrations and very-low-density lipoprotein (VLDL) levels in rats fed with 50 g/kg maitake were lowered compared with those of the control group (50 g/kg cellulose powder), and the fecal cholesterol excretion was significantly higher compared with the control group.

The antihypertensive effects of the active ingredients of maitake have been determined mainly through the measurement of systolic blood pressure (SBP) in animal models. An experiment was conducted on hypertensive rats with a diet containing 5% maitake or Lentinus edodes (L. edodes) mushroom powder. The results showed that both maitake and L. edodes treatment could significantly decrease the SBP of spontaneously hypertensive rats. The reduction in SBP level was similar for G. frodosa and L. edodes, which was around 15 mmHg after 63 days of a mushroom diet compared with the control group. Their further research showed that maitake could suppress the development of hypertension (preventive effect) and lower elevated blood pressure (treatment effect). Rats fed with two commercially-available fractions of SX and D were compared with a control group fed on a baseline diet and found that maitake fractions could lessen age-related hypertension partly via their effects on the renin-angiotensin system.

  1. Antioxidant Activities

Several components in maitake, including polysaccharides, proteins, fatty acids, and some other constituents, have shown notable antioxidant activities. The standard antioxidant activity assays include the scavenging abilities of hydroxyl radicals, DPPH radicals, superoxide radicals, and hydrogen peroxide, as well as the reducing power and Fe2+ chelating activity. Polysaccharides from maitake could be potential ingredients for cosmetic applications due to their antioxidant activity, radical scavenging activity after UV irradiation, the proliferation of fibroblasts, and collagen biosynthesis. Similar findings showed that the crude polysaccharide GFP, extracted from maitake fruiting bodies, possessed significant inhibitory effects on hydroxyl, superoxide, and DPPH radicals.

The antioxidant activity of maitake polysaccharides could be further enhanced by incorporating zinc or selenium. The strain of maitake was used as a vector of zinc biotransformation to produce zinc-incorporated intracellular polysaccharides, which showed notable antioxidant and anti-aging activities compared with the corresponding non-zinc-incorporated intracellular polysaccharides. Crude Se-polysaccharides (Se-GFP) from the fruiting bodies of Se-enriched maitake was purified to obtain a heteropolysaccharide of Se-GFP-22 with more remarkable antioxidant effects than that of non-Se-incorporated GFP-22. The antioxidant activity might be affected by the degree of branching, molecular weight, configuration, and the synergistic effect of polysaccharides and Se.

Other polysaccharides, proteins, fatty acids, and other molecules from maitake such as phenols and flavonoids, also showed antioxidant activity. Scientists hydrolyzed protein from the maitake fruiting body using different proteases. They found that trypsin hydrolysate had the strongest antioxidant potential, especially the GFHT-4 fraction, with a molecular weight lower than three kDa. Moreover, The inhibition levels of cyclooxygenase (COX)-1 enzyme and COX-2 enzyme activities by a fatty acid of maitake were 98% and 99%, respectively. Moreover, the inhibition of liposome peroxidation by the fatty acid was also as high as 79%. Several antioxidant components were obtained, including flavonoids, phenols, α-tocopherol, and ascorbic acid from the ethanol, cold-water, and hot-water extracts of maitake. These extracts exhibited various antioxidant activities, including reducing power, chelating ferrous ions, and scavenging DPPH and superoxide anions.

  1. Gut Microbiota Regulation

In recent years, there has been growing evidence of the important role of gut microbiota in the mediation/action of the various health benefits of mushrooms, especially their polysaccharide components. Many studies have investigated the regulation of gut microbiota by the bioactive polysaccharides from edible and medicinal mushrooms such as maitake because the biological macromolecules of polysaccharides cannot be directly absorbed by intestinal flora. The regulation of gut microbiota by polysaccharides was the major mechanism behind these properties. Specifically, maintaining gut microbiota homeostasis has been found to be related to improved treatment of type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). In vivo measurement of gut microbiota is usually preferred by high throughput sequencing to evaluate the microbiota regulation activity of bioactive components.

Researchers reported the regulatory efficacy of a novel maitake polysaccharide GFP-N on the intestinal microflora of diabetic groups in vivo using single-molecule real-time sequencing technology (SMRT). There were significant differences exhibited in the composition of microbial populations in gut microbiota between the GFP-N-treated group and the diabetic control group. The relative abundance of some bacterial species such as Lactobacillus acidophilus (L. acidophilus) and Bacteroides acidifaciens (B. acidifaciens) was increased with GFP-N treatment. L. acidophilus has been shown to delay the progression of high fructose-induced diabetes in rats, and B. acidifaciens has shown the potential for treating metabolic diseases such as obesity and diabetes. GFP could regulate intestinal microflora by significantly elevating the relative abundance of Alistipes and Bacteroides and reducing Enterococcus, which was associated with the improved hyperlipidemia and hyperglycemia in T2DM induced by streptozotocin and a high-fat diet (HFD). The same research group also developed maitake polysaccharide-chromium (III) (GFP-Cr(III)) through chelation because chromium (III) was the most important trace mineral for T2DM treatment. Compared with inorganic chromium, organic chromium (III) has been found to have much better effects, with lower toxicity and genotoxicity. The researchers found that GFP-Cr(III) not only had the effects of GFP as shown in their previous work but also significantly increased the relative abundance of Enterorhabdus and Coriobacteriaceae due to the presence of Cr(III).

GFP has also been found to regulate the gut microbiota of rats with non-alcoholic fatty liver disease (NAFLD). GFP could partly recover the HFD-induced alteration of cecal microbiota structure. GFP treatment could decrease the Firmicutes to Bacteroidetes ratio, indicating a lower possibility of lipid production from undigested carbohydrates. The ratio decrease of the two major gut bacteria classes, Firmicutes and Bacteroides, could have fat-lowering effects in obesity treatment. In addition, GFP supplementation significantly increased the proportion of Allobaculum, Bacteroides, Bifidobacterium, and other microbial groups in the cecal microbiota, which might boost the immune system of the host and the defense against NAFLD. The strengthening of the immune system may also contribute to GFP’s anti-tumor and anti-inflammatory effects. Nevertheless, the functions of gut microbiota on the various bioactivities of maitake polysaccharides and other components still require further exploration.