Bioactive Ingredients and Medicinal Values of Grifola frondosa (Maitake)

Grifola frondosa (G. frondosa), generally known as hen-of-the-woods or maitake in Japanese and hui-shu-hua in Chinese, is an edible mushroom with both nutritional and medicinal properties. This review provides an up-to-date and comprehensive summary of research findings on its bioactive constituents, potential health benefits and major structural characteristics. Since the discovery of the D-fraction more than three decades ago, many other polysaccharides, including β-glucans and heteroglycans, have been extracted from the G. frondosa fruiting body and fungal mycelium, which have shown significant antitumor and immunomodulatory activities. Another class of bioactive macromolecules in G. frondosa is composed of proteins and glycoproteins, which have shown antitumor, immunomodulation, antioxidant and other activities. A number of small organic molecules such as sterols and phenolic compounds have also been isolated from the fungus and have shown various bioactivities. It can be concluded that the G. frondosa mushroom provides a diverse array of bioactive molecules that are potentially valuable for nutraceutical and pharmaceutical applications. More investigation is needed to establish the structure–bioactivity relationship of G. frondosa and to elucidate the mechanisms of action behind its various bioactive and pharmacological effects.


Introduction
Grifola frondosa (G. frondosa) is a Basidiomycetes fungus that belongs to the family of Grifolaceae and the order of Polyporales. In Japan its edible fruiting body is known as maitake. In Japanese, mai means dance and take means mushroom. G. frondosa is known as "hui-shu-hua" (grey tree flower) in Chinese, possibly due to its appearance. G. frondosa grows around the stumps of broadleaf trees or trunks and is edible when young. The environment of the northeastern part of Japan is suitable for the growth of G. frondosa. The temperate forests in eastern North America, Europe and Asia are also ideal for its growth. Meanwhile, it is a common mushroom in the Unites States and Canada, known as sheep's head, king of mushrooms, hen-of-the-woods, and cloud mushroom [1].
Japan was one of the countries that first started the artificial cultivation of G. frondosa in the mid-1980s. There are in general three methods for the artificial cultivation of the G. frondosa fruiting body, they are bottle culture, bag culture and outdoor bed culture. Bag culture is the most popular cultivation method in Japan [2] because of its advantages such as the low cost of plastic bags, small space requirements and easily-controlled indoor environment. Bag culture can achieve higher yields of mature G. frondosa mushrooms than bottle culture and requires a shorter cultivation time than outdoor bed cultures. As shown in Figure 1 [2], the major steps of bag cultivation include substrate preparation, substrate sterilization, mycelium inoculation and incubation. In addition to the fruiting body, there is also an increasing demand for G. frondosa's mycelium and its bioactive metabolites. Solidstate fermentation (SSF) [3] and submerged fermentation [4] are two common methods of mycelium cultivation. A common substrate for SSF is sawdust supplemented with rice bran is also an increasing demand for G. frondosa's mycelium and its bioactive metabolites. Solid-state fermentation (SSF) [3] and submerged fermentation [4] are two common methods of mycelium cultivation. A common substrate for SSF is sawdust supplemented with rice bran or wheat bran [5]. Submerged or liquid fermentation is usually more efficient, providing a higher mycelial productivity in a shorter time, requiring smaller plant space and allowing for more effective product quality control [6]. A typical submerged fermentation process is presented in Figure 2.   [4].
G. frondosa is edible and is regarded as a healthy food because it is a good source of protein, carbohydrates, dietary fiber [7][8][9][10][11][12][13], vitamin D2 (ergocalciferol) [13][14][15] and minerals (K, P, Na, Ca, Mg) [7,9,12,15,16], with low fat content and caloric value [15]. G. frondosa is delicious, with a sweet and umami taste, which is mainly attributed to its high trehalose, glutamic and aspartic amino acid and 5′-nucleotide content [10,11,13,17]. Due to its delicious and special taste, G. frondosa is not only used as a food ingredient, but also as a food-flavoring substance in dried powder form. Apart from its high nutraceutical  is also an increasing demand for G. frondosa's mycelium and its bioactive metabolites. Solid-state fermentation (SSF) [3] and submerged fermentation [4] are two common methods of mycelium cultivation. A common substrate for SSF is sawdust supplemented with rice bran or wheat bran [5]. Submerged or liquid fermentation is usually more efficient, providing a higher mycelial productivity in a shorter time, requiring smaller plant space and allowing for more effective product quality control [6]. A typical submerged fermentation process is presented in Figure 2.   [4].
G. frondosa is edible and is regarded as a healthy food because it is a good source of protein, carbohydrates, dietary fiber [7][8][9][10][11][12][13], vitamin D2 (ergocalciferol) [13][14][15] and minerals (K, P, Na, Ca, Mg) [7,9,12,15,16], with low fat content and caloric value [15]. G. frondosa is delicious, with a sweet and umami taste, which is mainly attributed to its high trehalose, glutamic and aspartic amino acid and 5′-nucleotide content [10,11,13,17]. Due to its delicious and special taste, G. frondosa is not only used as a food ingredient, but also as a food-flavoring substance in dried powder form. Apart from its high nutraceutical Generally, proximate composition is determined by the methods suggested by the Association of Official Analytical Chemists (AOAC). The total carbohydrate content can be calculated by subtracting the percentages of ash, crude fat and protein [7,40]. For the determination of crude protein, the nitrogen conversion factor is 4.38 instead of the usual 6.25, due to the large amount of chitin that is usually contained within the fungus, a component that may interfere with the correct calculation of the result of total nitrogen [41].
As shown in Table 1, G. frondosa is made up of around 83-96% moisture and 4-17% dry matter in its fresh fruiting body [7][8][9][10][11][12][13] and mycelium [11,17,42], indicating the watery texture of G. frondosa. Carbohydrates and protein are the major constituents contributing to the dry weight of G. frondosa, taking up around 70-80% and 13-21%, respectively, of the fruiting body. Based on the average values of component percentage, it could be found that the mycelium of G. frondosa has a similar moisture content, a lower content of carbohydrate and crude ash and a higher content of crude fat and protein, compared with the fruiting body of G. frondosa. According to the research findings of Kurasawa and coworkers [8], the composition of the G. frondosa fruiting body resembles that of normal cultivated mushrooms. It is worth mentioning that the crude fat content of the G. frondosa fruiting body is generally lower than the average crude fat content in cultivated mushrooms (4.3%), and the amounts of protein and carbohydrates are slightly higher than the average of other mushrooms (17.2% and 70.3%), indicating the excellent nutritional values of G. frondosa.

Soluble Sugar Content
The content of soluble sugar within G. frondosa is mostly determined by the method described in the research work of Ajlouni and coworkers [42]. As shown in Table 2, the total sugar content in G. frondosa is higher in the fruiting body (90-190 mg/g) than in the mycelium (70-90 mg/g). The content of total sugar in the fruiting body of G. frondosa is also superior to some edible mushrooms such as Lactarius glaucescens and Craterellus odoratus [45], which may be one of the reasons for the good taste of G. frondosa. Table 2 also shows variations in both total soluble sugar content and individual sugar content among different G. frondosa samples, which may be attributed to factors such as cultivation period and cultivation environment. Trehalose, a disaccharide that comprises two molecules of glucose, is the major sugar component of both the fruiting body and mycelia of G. frondosa [9][10][11]43,44]. Compared with the amount in the mycelium (40-60 mg/g), the fruiting body contains more trehalose than the mycelium, around 50-160 mg/g in dry weight. In addition to trehalose, the fruiting body also contains glucose and mannitol, whereas the mycelium has glucose and mannitol, together with arabitol and fructose.

Free Amino Acid Content
The content of free amino acids in G. frondosa was quantitatively measured by the method described in the research work of Mau and coworkers using HPLC [10,44]. Results regarding the free amino acid content of G. frondosa are exhibited in Table 3. The total free amino acid content in the fruiting body of G. frondosa is around 15-60 mg/g in dry weight, which is higher than that in many other edible mushrooms, such as Dictyophora indusiata and Tricholoma giganteum [10]. The mycelium of G. frondosa contains a relatively higher total free amino acid content in comparison with the fruiting body. There is also a great variety of amino acids in G. frondosa. There are around eighteen kinds of free amino acids, including essential amino acids such as L-histidine and L-methionine, in both the fruiting body and the mycelium of G. frondosa, indicating that G. frondosa is an excellent source of amino acids. However, large variations exist in the amount of each amino acid in G. frondosa. For instance, Mau et al. (2001) and Tsai et al. (2006) found that threonine was the major free amino acid in G. frondosa [10,44], whereas Huang and coworkers found that histidine and glutamic acid were the major free amino acids in the fruiting body (>12 mg/g) and lysine, aspartic acid and tyrosine were the major free amino acids in the mycelium (>17 mg/g) [11]. Huang and coworkers also obtained a larger amount of free amino acid from G. frondosa than other groups in both the fruiting body and the mycelium, a result that might be due to the different sources of the fruiting body and the preparation methods of the mycelium by the different research groups. The choice in cultivation substrate was also found to affect the variety and amount of amino acids in G. frondosa. As shown in Table 3, G. frondosa fruiting bodies cultivated in sawdust and log substrates have different amounts of each amino acid [13], although the total amino acid content was similar. Moreover, GABA (γaminobutyric acid), a biologically active compound which is related to the therapeutic effect of G. frondosa, is mainly detected in the mycelium but not in the fruiting body [11] (Table 3).

Polysaccharides
In the past 30 years, over 47 bioactive polysaccharide fractions have been isolated and purified from the fruiting body, mycelium and cultured medium of G. frondosa using different extraction methods. As previously reported, G. frondosa contains 3.8% watersoluble polysaccharides on a dry weight basis, of which 13.2% was (1→3, 1→6)-β-Dglucan [46], and others include heteroglycan or the heteroglycan/protein complex [47]. Among these bioactive polysaccharide fractions, the D-fraction and the MD-fraction (purified D-fraction) [22], which are regarded as the most important bioactive polysaccharides, have been officially used as antitumor, anticancer and immunomodulatory agents [48]. Through structural characterization, Nanba and Kubo discovered the complex structure of the β-D-glucan in the D-fraction. Unlike other mushroom-derived β-glucans that contained a (1→3) main chain with (1→6) branches only, the β-D-glucan in D-fraction possessed both a (1→6) main chain with (1→3) branches and a (1→3) main chain with (1→6) branches [22]. The high molecular weight of the D-fraction was considered a factor contributing to its strong immunomodulatory effects [22]. Figure 3 shows a typical structure of the D-fraction. The D-fraction and the MD-fraction could be extracted and fractionated from both the mycelium and fruiting body of G. frondosa. Figure 4 is a general flowchart for the extraction and purification of the MD-fraction as reported by Nanba and coworkers. The MD fraction is typically extracted from the dried G. frondosa powder with boiling water and then isolated by ethanol precipitation. The precipitate (crude MD fraction) is further fractionated through column chromatography, including ion exchange and gel permeation chromatography, to obtain the purified MD fraction.
In the past 30 years, over 47 bioactive polysaccharide frac and purified from the fruiting body, mycelium and cultured med different extraction methods. As previously reported, G. frondosa uble polysaccharides on a dry weight basis, of which 13.2% was [46], and others include heteroglycan or the heteroglycan/prote these bioactive polysaccharide fractions, the D-fraction and the M fraction) [22], which are regarded as the most important bioactiv been officially used as antitumor, anticancer and immunom Through structural characterization, Nanba and Kubo discovere of the β-D-glucan in the D-fraction. Unlike other mushroom-der tained a (1→3) main chain with (1→6) branches only, the β-D-g sessed both a (1→6) main chain with (1→3) branches and a (1→3 branches [22]. The high molecular weight of the D-fraction was tributing to its strong immunomodulatory effects [22]. Figure 3 s of the D-fraction. The D-fraction and the MD-fraction could be ex from both the mycelium and fruiting body of G. frondosa. Figure for the extraction and purification of the MD-fraction as reported ers. The MD fraction is typically extracted from the dried G. frond water and then isolated by ethanol precipitation. The precipitat further fractionated through column chromatography, includin permeation chromatography, to obtain the purified MD fraction  Apart from the D-fraction and the MD-fraction, other polysaccharide fractions have also been derived from G. frondosa, with hot water being the most commonly used extraction solvent. Ultrasound [51] and other solvents such as hot sodium hydroxide [18] and citrate buffer [24] have also been utilized. Table 4 summarizes the chemical properties, sources and extraction solvents of representative bioactive polysaccharide fractions isolated from G. frondosa. The chemical properties listed include molecular weight, structure information, as well as monosaccharide compositions. For instance, Kubo et al. extracted the X-fraction from the fruiting body of G. frondosa, which was a (1→6)-β-glucan with (1→4)-α branches. The X-fraction showed anti-diabetic activity, which was found to be directly associated with insulin receptors [23]. Masuda et al. separated the MZ-fraction from G. frondosa, which had similar structure to the MD-fraction but with a much smaller molecular weight, of around 20,000 Da (the molecular weight of MD-fraction was around 1-2 million Da). The MZfraction showed immunomodulatory effects in vitro and antitumor activity in vivo [25]. Moreover, Grifolan (GRN), a gel-forming (1→6)-branched (1→3)-β-D-glucan, was found in G. frondosa with immunomodulatory effects [24]. α-D-glucan could also be extracted by hot water from G. frondosa. Instead of antitumor activity, hypoglycemic, hypolipidemic, antioxidative and immunomodulatory effects were discovered [26,[52][53][54]. In recent years, Liu's group from China has isolated various polysaccharides from G. frondosa with the names of GFP-A [55], LMw-GFP [51] and GFAP [56], all of which exhibited promising anti-tumor activities. Due to variations in external factors such as the fungal source and extraction temperature, the properties of polysaccharide fractions from different sources may vary significantly.

Figure 4. A typical extraction method of the MD fraction from G. frondo
Apart from the D-fraction and the MD-fraction, other polys also been derived from G. frondosa, with hot water being the mos tion solvent. Ultrasound [51] and other solvents such as hot sod citrate buffer [24] have also been utilized. Table 4 summarizes sources and extraction solvents of representative bioactive poly lated from G. frondosa. The chemical properties listed include mo information, as well as monosaccharide compositions. For instan the X-fraction from the fruiting body of G. frondosa, which wa (1→4)-α branches. The X-fraction showed anti-diabetic activity, directly associated with insulin receptors [23]. Masuda et al. se from G. frondosa, which had similar structure to the MD-fraction molecular weight, of around 20,000 Da (the molecular weight of 1-2 million Da). The MZ-fraction showed immunomodulatory tumor activity in vivo [25]. Moreover, Grifolan (GRN), a gel-f

Proteins and Peptides
Several kinds of bioactive proteins and peptides have been isolated from G. frondosa with notable health benefits and medicinal values, although studies on these aspects are fewer than those on the bioactive polysaccharide fractions. Table 5 summarizes some typical bioactive proteins and peptides isolated from G. frondosa. As shown in the table, these bioactive proteins/peptides were extracted mainly from the G. frondosa fruiting body, with average molecular weights around 20-88 kDa. The N-acetylgalactosamine-specific lectin GFL isolated from the G. frondosa fruiting body exhibited cytotoxicity against HeLa cells [71]. The protein designated GFAHP showed a significant anti-herpes simplex virus (HSV) effect, as reported by Gu and coworkers [35]. Glycoprotein is another type of bioactive protein in G. frondosa. Cui and coworkers isolated the glycoprotein (containing 6.2% carbohydrates) from cultured mycelia of G. frondosa and demonstrated its anti-tumor activity [32]. Zhuang and coworkers patented a bioactive glycoprotein from G. frondosa, which showed obvious anti-hypertensive, antiobesity, anti-hyperlipidemic and anti-diabetic effects [34]. In addition to the glycoprotein, Chan et al. found that chemical phosphorylation of G. frondosa polysaccharide-peptides could remarkably enhance both tumor inhibition in vitro and adjuvant effects in vivo. Meanwhile, modified and unmodified MPSP both showed an insignificant effect on normal cells, indicating their potential application for anticancer therapy without significant side effects [72].

Other Bioactive Molecules
Apart from the macromolecular components, such as polysaccharides and proteins/ peptides, bioactive small molecules have also been discovered in G. frondosa. Table 6 lists some bioactive small molecules in G. frondosa from representative research studies. The major small molecules discovered with bioactivities mainly include fatty acids, ergosterols, flavonoids, alkaloids, ascorbic acid and tocopherol. AGF -Acetone, mycelium [36] Grifolaone A Ethyl acetate, mycelium [74] GF-3 Pyrrolefronine, seven pyrrole alkaloids and nine ergosterols Ethanol, fruiting body [37] Ergosterol peroxide -Methanol, fruiting body [75] As reported by Zhang and coworkers, fatty acids and three compounds, namely ergosterol (1), ergostra-4,6,8(14),22-tetraen-3-one (2) and 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (3), were extracted from the cultured mycelia of G. frondosa by hexane. The fatty acid fraction, together with all three compounds, exhibited cyclooxygenase (COX) enzyme inhibitory and anti-oxidant activities [73]. He and coworkers extracted a furanone named grifolaone A from G. frondosa, which showed specific antifungal activity against the opportunistic human pathogen of Pseudallescheria boydii and some plant pathogens [74]. Han and Cui isolated agaricoglycerides (AGF) from the fermented mycelium of G. frondosa. Their study suggested a promising possibility of using AGF as medicine for inflammatory pain with 500 mg/kg as the optimal dosage [36]. Chen et al. extracted three fractions, GF-1 to GF-3, from G. frondosa and discovered the inhibitory effect of GF-3 against the proliferation of human tumor cells and α-glucosidase. The major bioactive compounds in GF-3 were detected to be alkaloids (first found in G. frondosa), ergosterols and a new compound named pyrrolefronine. Since α-glucosidase was involved in the hydrolysis of starch into disaccharide sugars, inhibition of its activity indicated possible reduction of blood glucose level [37]. o-orsellinaldehyde, which showed obvious tumoricidal activity, especially selective cytotoxic effect against Hep 3B cells through apoptosis, was also extracted from a submerged culture of G. frondosa by Lin and Liu [38]. In addition, other bioactive molecules in G. frondosa, such as polyphenolics, α-tocopherol, ascorbic acid and flavonoids, were reported to have anti-oxidant properties [39].

Antitumor Effects
Previous studies over the past 30 years have strongly suggested that there are three possible ways by which G. frondosa exerts its anticancer effect-they are protection of healthy cells, prevention of tumor metastasis and inhibition of tumor growth. In other words, G. frondosa can fight against tumors both directly and indirectly via enhancement of the immune system. This section will mainly focus on the direct antitumor function of G. frondosa, whereas the immunomodulatory effect will be discussed in the next section.
The antitumor activity of G. frondosa was first reported by Miyazaki et al. in 1982 [16], followed by their further study on the chemical structure of glucans extracted from the G. frondosa fruiting body and their antitumor activity against Sarcoma 180 tumors in Institute of Cancer Research (ICR) mice [17]. Nanba's group obtained different polysaccharide fractions and reported the D-fraction for the first time in 1988 [21]. 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 [21]. Nanba and Kubo conducted a nonrandomized clinical study to assess the effects of the D-fraction from G. frondosa on 165 advanced cancer patients who received the D-fraction as crude powder tablets alone or in addition to chemotherapy. Results showed that G. frondosa was effective against breast, liver and lung cancers, but less effective against leukemia, stomach and bone cancers [22]. Further studies conducted by Alonso et al. demonstrated that the D-fraction was able to function on mammary tumor cells directly through the modulation of different cellular processes during cancer development [48]. Zhao and coworkers found that a combination of the D-fraction (0.2 mg/mL) and vitamin C (0.3 mmol/L) resulted in a 70% reduction in the viability of human hepatocarcinoma SMMC-7721 cells [76]. Further purification of the D-fraction yielded the MD-fraction, which, as described in the patent of Nanba and Kubo, showed even better results than the D-fraction in terms of the inhibitory effect on mouse tumor growth [22]. In addition to intraperitoneal injection in Nanba and Kubo's test, the MD-fraction has also been demonstrated to inhibit tumor growth in mice via oral administration [77]. Both the D-fraction and the MD-fraction were proven safe, with low or no toxicity.
Apart from the D and MD-fractions, other polysaccharide fractions have also exhibited anti-tumor activity. As reported by Bie et al. [65], the polysaccharide GFP-A, isolated from G. frondosa, was able to inhibit the proliferation of human colon cancer HT-29 cells in vitro, with 180 µg/mL as the most effective concentration. Li and Liu reported that the polysaccharide fraction GFP-4, extracted from G. frondosa, showed an inhibitory effect on human lung cancer cells at 4 • C. The inhibitory effect became lower after heat treatment at over 30 • C due to structural changes [78]. Alonso and coworkers explained that the polysaccharides in G. frondosa could regulate gene expression involved in the apoptosis of breast cancer cells so that cell proliferation was inhibited and the cell cycle was blocked [79].
In addition to polysaccharide fractions, the ergosterol derivatives from non-polar extracts of G. frondosa were also found to have anti-proliferative effects on human tumor cells [37]. Moreover, the o-orsellinaldehyde component of submerged cultures of G. frondosa exhibited tumoricidal activity against Hep 3B cells via apoptosis [38]. Some glycoproteins isolated from G. frondosa, such as GFL and GFG-3a, exhibited antitumor effects as well due to their anti-proliferative activity against cancer cells [32,71]. Table 7 summarizes the testing methods and potency of antitumor activity based on reported studies on G. frondosa. The testing methods for in vivo studies mainly include microscopy observation and assessment of the inhibition rate by measuring tumor weight, whereas for in vitro studies, the MTT assay is commonly used to determine cells' viability. It is worth mentioning that G. frondosa was able to achieve an inhibition ratio of over 90% for the treatment of MM46 liver carcinoma, BEL 7402 cells and TMK-1 gastric cancer cells.

PS GFP
In vivo measurement of gut microbiota in high-fat-diet-fed rats by high-throughput sequencing For GFP (400 mg/kg day)-treated group, significant increase in the relative abundance of Helicobater, Intestinimonas, Barnesiella, Parasutterella, Ruminococcus and Flavonifracter, and decrease in Clostridium-XVIII, Butyricicoccus and Turicibacter. Similar gut microbiota composition to that of the normal group.

PS GFP
In vivo access of gut microbiota in high-fat diet-fed and streptozotocin-treated mice by high throughput sequencing.
For GFP-treated (900 mg/kg day) group, significant increase in the relative abundance of Alistipes and Bacteroides, and decrease in Enterococcus. [96]

PS GFP
In vivo evaluation of gut microbiota in high-fat-diet-fed rats by high-throughput next-generation 16S rRNA gene sequencing.
For GFP-treated (150 mg/kg day) group, significant increase in the relative abundance of Allobaculum, Bacteroides, Bifidobacterium and other cecal microbiota compared with the HFD-fed group. [30]

PS GFWE
In vivo access of gut microbiota in high-sucrose-and high-fat-diet-fed rats by real-time sequencing.

Immunomodulation
Immunomodulation is the most well-known effect of G. frondosa 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 [99]. Furthermore, G. frondosa 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 G. frondosa. The D-fraction, in addition to its direct antitumor effect as mentioned previously, is also a major polysaccharide fraction of G. frondosa with significant immunomodulatory activity. Kodama and coworkers suggested that the D-fraction could activate NK cells through upregulating their expression of TNF-α and interferon-gamma (IFN-γ) proteins. Meanwhile, the D-fraction were also able increase macrophage-derived IL-12, which further activated NK cells, implying that the D-fraction could provide long-term tumor-suppressive effects [49,82]. Further investigation by Kodama et al. found that 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 [80].
Apart from the D-fraction, other polysaccharide fractions with immunomodulatory activity have also been isolated from G. frondosa. Ishibashi and coworkers isolated insoluble and a high-molecular-weight soluble forms of Grifolan (GRN) from G. frondosa, both of which can activate macrophages through triggering cytokine secretion to produce TNF [61,100]. 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 G. frondosa, suggesting the activation of macrophages and the stimulation of tumoricidal activity [64]. Masuda and coworkers found that the anti-cancer activity of the polysaccharide fraction MZF from G. frondosa 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 [25,59]. The GFP fraction isolated by Meng et al. 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 strong immune-stimulating activity [68]. Table 7 presents the common testing methods and potency indices of immunomodulatory effects. In vitro testing is usually performed with cytokine production evaluated based on ELISA and macrophage activity by MTT assay.

Antiviral and Antibacterial Effects
There have been a number of studies reporting the beneficial effects of G. frondosa in the treatment of viral infections, including those caused by hepatitis B virus (HBV), enterovirus 71 (EV71), herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV). Mayell and coworkers reported a study on patients with chronic hepatitis B. The results showed that patients who took G. frondosa fruiting body polysaccharides showed positive signs, specifically a higher recovery rate compared with the control group [1]. Nanba et al. reported that the MD-fraction from G. frondosa could fight against HIV through several pathways, including direct inhibition of HIV, stimulation of the natural defense system against HIV and a reduction in vulnerability to opportunistic infections [50]. The GFP1 fraction extracted by Zhao and coworkers was found to fight against EV71, the causative pathogen of hand-foot-and-mouth disease. The researchers found that G. frondosa 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 [27]. In addition to polysaccharide fractions, the protein It significantly inhibited HSV-1 replication in vitro and reduced HSV-1 induced symptoms such as blepharitis in a murine model [35].
In addition to antiviral effect, the D-fraction isolated from G. frondosa has also shown antibacterial effects. Kodama and coworkers found that 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 [84]. Unlike the antibacterial mechanism, the antiviral action of the D-fraction is not directly related to the immune system. According to Gu et al., the D-fraction interfered with HBV replication through the inhibition of HBV polymerase [85].

Antidiabetic Activity
The hypoglycemic effects of G. frondosa extracts have been demonstrated in multiple animal studies. To test the antidiabetic activity of active ingredients in G. frondosa, 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 ( Table 7). A high FSG level is one of the characteristics of diabetes mellitus sufferers and the influence on FSG level could directly indicate the antidiabetic effect of active ingredients in G. frondosa.
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 hypoglycemic effect through insulin signal pathway [63,87]. Konno et al. reported that the SX-fraction could facilitate glucose uptake, leading to activation of the insulin receptor (IR) and insulin receptor substrate 1 (IRS-1), and 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 [87]. The hypoglycemic mechanisms of F2 and F3 polysaccharides were also related to IR and IRS-1. Xiao et al. reported that they could improve insulin sensitivity and decrease FSG levels by increasing protein levels of IR and decreasing protein levels of IRS-1 [63]. 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 [26].
In addition to the enhancement of insulin activity, the hypoglycemic effects of G. frondosa may be generated through the inhibition of α-glucosidase activity, because an antiα-glucosidase effect could prevent starch hydrolysis into disaccharides and decrease the blood glucose level. Shen and coworkers examined the hyperglycemic effects of non-polar fractions in G. frondosa both in vivo and in vitro. Research findings showed that G. frondosa 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 [86]. Chen et al. attributed the anti-α-glucosidase effect to the pyrrole alkaloids and ergosterols extracted from G. frondosa [37], whereas Wu et al. suggested that the ergosterol peroxide isolated from G. frondosa contributed to its anti-diabetic effect [75]. However, Su and coworkers concluded that the strong anti-α-glucosidase activity was mainly attributed to the oleic acid and linoleic acid, rather than ergosterol and ergosterol peroxide in G. frondosa [101]. Some previous studies suggested that the anti-diabetic activity of G. frondosa originated from its regulatory effect on gut microbiota, which shall be discussed later in this paper.

Lipid Metabolism Regulation and Anti-Hypertension Effects
The effects of G. frondosa on lipid metabolism regulation and anti-hypertension have been shown in many reports. Kubo and Nanba found that with the G. frondosa fruiting body as the feed, the triglyceride, cholesterol and phospholipid levels in the serum of rats were 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, and the corresponding cholesterol excretion ratio in feces increased by 1.8 times with G. frondosa treatment, further demonstrating that G. frondosa treatment helped improve lipid metabolism and inhibit increases in liver lipid and serum lipid after the ingestion of high-fat feed [88]. Similar results were obtained by Fukushima and coworkers, who showed that serum total cholesterol concentrations and very-low-density lipoprotein (VLDL) levels in rats fed with 50 g/kg G. frondosa 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 [89].
The antihypertensive effects of the active ingredients of G. frondosa have been determined mainly through the measurement of systolic blood pressure (SBP) in animal models, as summarized in Table 7. Kabir et al. conducted an experiment on hypertensive rats with a diet containing 5% G. frondosa or Lentinus edodes (L. edodes) mushroom powder. The results showed that both G. frondosa and L. edodes treatment could result in a significant decrease in 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 [102]. Their further research showed that G. frondosa could not only suppress the development of hypertension (preventive effect), but also lower elevated blood pressure (treatment effect) [103]. Preuss and coworkers compared rats fed with two commercially-available fractions of SX and D with a control group fed on a baseline diet and found that G. frondosa fractions could lessen age-related hypertension partly via their effects on the renin-angiotensin system [90].

Antioxidant Activities
Several components in G. frondosa, including polysaccharides, proteins, fatty acids and some other constituents, have shown notable antioxidant activities. The common activity assay methods and anti-oxidant potencies are summarized in Table 7. As shown in this table, the common antioxidant activity assays include the scavenging abilities of hydroxyl radicals, DPPH radicals, superoxide radicals and hydrogen peroxide; as well as the reducing power and Fe 2+ chelating activity. Lee and coworkers suggested that polysaccharides from G. frondosa could be potential ingredients for cosmetic applications due to their antioxidant activity, radical scavenging activity after UV irradiation, proliferation of fibroblasts and collagen biosynthesis [92]. Similar findings were obtained by Chen and coworkers, who showed that the crude polysaccharide GFP, extracted from G. frondosa fruiting bodies, possessed significant inhibitory effects on hydroxyl, superoxide and DPPH radicals [93].
The antioxidant activity of G. frondosa polysaccharides could be further enhanced by incorporation of zinc or selenium. Zhang and coworkers used the strain of G. frondosa 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 [91]. Li and coworkers purified crude Se-polysaccharides (Se-GFP) from the fruiting bodies of Se-enriched G. frondosa and obtained a heteropolysaccaride 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 and configuration, as well as the synergistic effect of polysaccharide and Se [67].
Other than polysaccharides, proteins, fatty acids and other molecules from G. frondosa such as phenols and flavonoids also showed antioxidant activities. Dong and coworkers hydrolyzed protein from the G. frondosa fruiting body using different proteases and found that trypsin hydrolysate had the strongest antioxidant potential, especially the GFHT-4 fraction, with a molecular weight lower than 3 kDa [94]. Moreover, according to Zhang et al., the inhibition levels of cyclooxygenase (COX)-1 enzyme and COX-2 enzyme activities by a fatty acid of G. frondosa were 98% and 99%, respectively. Moreover, the inhibition of liposome peroxidation by the fatty acid was also as high as 79% [73]. Yeh and coworkers obtained several antioxidant components including flavonoids, phenols, α-tocopherol and ascorbic acid from the ethanol, cold-water and hot-water extracts of G. frondosa. All of these extracts exhibited various antioxidant activities, including reducing power, chelating ferrous ions and scavenging DPPH and superoxide anions [39].

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 [104]. Many studies have investigated the regulation of gut microbiota by the bioactive polysaccharides from edible and medicinal mushrooms such as G. frondosa because the biological macromolecules of polysaccharides, which cannot be directly absorbed, can be utilized by intestinal flora [105]. Friedman reviewed mushroom polysaccharides and their anti-obesity, anti-diabetes, anti-cancer and antibiotic properties, and suggested that the regulation of gut microbiota by polysaccharides was the major mechanism behind these properties [106]. Specifically, the maintaining of gut microbiota homeostasis has been found to be related with improved treatment of type 2 diabetes mellitus (T2DM) [107] and non-alcoholic fatty liver disease (NAFLD) [108]. To evaluate the microbiota regulation activity of bioactive components, in vivo measurement of gut microbiota is usually preferred by high throughput sequencing, as shown in Table 7.
Very recently, Chen and coworkers reported the regulatory efficacy of a novel G. frondosa polysaccharide GFP-N on the intestinal microflora of diabetic groups in vivo using single-molecule real-time sequencing technology (SMRT) [31]. 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 [96], and B. acidifaciens has shown the potential for treatment of metabolic diseases such as obesity and diabetes [109]. Guo et al. obtained similar results, showing that 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) [107]. The same research group also developed G. frondosa 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) [110].
Additionally, GFP has also been found to regulate the gut microbiota of rats with non-alcoholic fatty liver disease (NAFLD). Li and coworkers found that GFP could partly recover the HFD-induced alteration of cecal microbiota structure [30]. GFP treatment could decrease the Firmicutes to Bacteroidetes ratio, indicating a lower possibility of lipid production from undigested carbohydrates [108]. Friedman also suggested in his review that the ratio decrease of the two major classes of gut bacteria, namely, Firmicutes and Bacteroides, could have fat-lowering effects in obesity treatment [106]. In addition, GFP supplementation significantly increased the proportion of Allobaculum, Bacteroides, Bifidobacterium and some other microbial groups in the cecal microbiota, which might boost the immune system of the host and the defense against NAFLD [30]. The boosting of the immune system may contribute to the anti-tumor and anti-inflammatory effect of GFP as well [29,111]. Nevertheless, the functions of gut microbiota on the various bioactivities of G. frondosa polysaccharides and other components still require further exploration.

Conclusions
Edible and medicinal fungi or mushrooms are among the most common sources of health foods and nutraceutical products. G. frondosa is one of the most widely explored fungal species for nutraceutical and therapeutic compounds. The fungal biomass of G. frondosa displays a high content of proteins and carbohydrates and a relatively low content of fat compared with other commonly cultivated mushrooms. The crude water extracts, isolated fractions and purified components have shown a number of bioactivities, including antitumor, immunomodulatory, antiviral, antibacterial, antidiabetic, lipid metabolism regulation, hypertension control and antioxidation. Some of these health effects may be associated with the regulation of the human gut microbiota. Polysaccharides, which represent the most significant bioactive components of G. frondosa, contribute to many of its bioactivities and health benefits. The most successful and valuable health products from this fungal species are represented by the polysaccharide fractions and polysaccharide protein complexes, including the D-fraction or the MD-fraction and Grifolan, which have been approved for human use in immunotherapy and complimentary treatment of cancers with chemotherapy and radiotherapy.
Although some of the constituents of G. frondosa have been widely used in health foods or dietary supplements, very few have been used in prescribed medication, which requires more rigorous assessment and clinical trials. For a wider and more reliable application of the various components in nutraceutical and therapeutic products, it is fundamental to gain a better understanding of the structure-bioactivity relationship and the underlying mechanisms of action in the human body. Structural modification of the polysaccharides is another feasible strategy to attain enhanced bioactivity and novel bioactive molecules. As for many food and medicinal products, good manufacturing practice (GMP) should be implemented in the production process and systems, and standardized protocols should be established and followed for the preparation and quality control of the useful components.
With increasing public concern about health threats from food contamination, environmental pollution and new infectious organisms such as the COVID-19 virus, the protection of human health through the immunomodulatory and health-promoting functions of G. frondosa constituents is even more attractive and promising. Therefore, it is worthwhile to put more effort into the research and development of this edible fungal species.