Download

REVIEW ARTICLE

Extraction, isolation, identification, and bioactivity of polysaccharides from Antrodia cinnamomea

Jia-ning Dai1#, Bo-ling Liu1#, Dan Ji1, Lei Yuan1,2, Wen-yuan Zhou1, Hua-xiang Li1*

1School of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu, China;

2Key Laboratory of Zoonoses, Yangzhou, Jiangsu, China

#These authors contributed equally to this study and share first authorship.

Abstract

Antrodia (A) cinnamomea is a precious edible and medicinal mushroom and has attracted attention because of its rare resources and unique bioactivities. The bioactive compounds from the fruit body, mycelium, and fermentation broth of A. cinnamomea include triterpenoids, polysaccharides, antroquinonols, benzenoids, and succinic and maleic acid derivatives. Among these, polysaccharides are very important and are high-content bioactive compounds. The A. cinnamomea polysaccharides (APSs) present numerous biological activities, such as antiviral, anticancer, anti-inflammatory, antivascular, immunoregulation, antioxidant, and nerve protection. However, only few studies have focused only on APSs so far. Therefore, the cultivation methods, extraction, isolation, composition, structure, biological activity, and application of APSs are summarized in this review to provide a comprehensive and convenient reference for further research and development on APSs.

Key words: Antrodia cinnamomea, bioactivity, composition, extraction, isolation, polysaccharides

*Corresponding Author: Hua-xiang Li, School of Food Science and Engineering, Yangzhou University, Yangzhou, Jiangsu, China. Email: [email protected]

Received: 21 June 2023; Accepted: 9 October 2023; Published: 27 October 2023

DOI: 10.15586/qas.v15i4.1341

© 2023 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

The edible fungi can be used as food as well as medication. A number of edible fungi products have been identified as sources of healthy food supplements and drugs for numerous types of cancers in humans (Hsieh et al., 2006; Lavi et al., 2006; Lee et al., 2005), and omics techniques have been used to investigate active ingredients obtained from edible fungi (Cao et al., 2023; Geng et al., 2022). A variety of bioactive components, such as terpenes, polysaccharides, maleic acids, and their derivatives, have been identified from different types of edible fungi having good therapeutics, such as anticancer, antitumor, anti--inflammatory, hypoglycemic, hypotensive, and immunoregulatory properties (Cao et al., 2023; Geng et al., 2022; Lavi et al., 2006; Lee et al., 2005). Polysaccharides are considered as the most promising pharmacologically active components (Lavi et al., 2006). The polysaccharide (PS) of Astragalus membranaceus has excellent anticancer potential (Yang et al., 2013) and can alleviate obesity, liver steatosis, neuroinflammation, and cognitive impairment (Huang et al., 2017). Polysaccharides (PSs) obtained from several mushrooms, such as Ganoderma lucidum, Grifola umbellata, and Coriolus versicolor, scavenge free radicals (Liu et al., 1997) and inhibit tumor growth (Yukawa et al., 2012).

Antrodia cinnamomea (A. cinnamomea), with a common name of Antrodia camphorata, is a precious edible and medicinal mushroom that belongs to the phylum Basidiomycetes, family Polyporaceae, and genus Antrodia. It features a sporangium on the surface of the fruiting body that contains spores, an uneven surface, and a safrole odor. Based on the color of fruiting -bodies, the fungi are classified as red, yellow, and white A. cinnamomea (Su, et al., 2023). Triterpenoids and polysaccharides are the primary active components of A. cinnamomea (He et al., 2019; Liu et al., 2004). However, the wild fruiting body of A. cinnamomea is scarce and expensive and cannot meet market demand. Thus, the artificial cultivation of A. cinnamomea has become necessary. The main artificial cultivation methods of A. cinnamomea include basswood culture, plate culture, solid-state fermentation, and submerged fermentation (Li et al., 2015; Lu et al., 2014b). Submerged fermentation is the most applied cultivation method because of its short period, high production efficiency, and easy to scale-up production (Zhang et al., 2019).

Dr. Huaxiang Li is engaged in the research on A. cinnamomea for more than 10 years, especially focusing on the submerged fermentation of A. cinnamomea. In 2009, it was reported for the first time that A. cinnamomea could produce a large number of asexual spores (i.e., arthrospores) at a late stage of submerged fermentation under appropriate environmental and nutritional conditions (Lu et al., 2011). Then, in 2014, the rapid fermentation process of A. cinnamomea based on asexual spore inoculation was established for the first time (Lu et al., 2014b). In 2015, to shorten further the production period and save production cost, an efficient repeated batch fermentation process based on asexual spore inoculation was established, which greatly improved the production efficiency of A. cinnamomea in submerged fermentation (Li et al., 2015). In 2017, proteomics and transcriptome techniques were used to explore the molecular regulatory mechanism underlying the asexual sporulation of A. cinnamomea during submerged fermentation and successfully revealed the FluG-mediated asexual sporulation signaling pathway in A. cinnamomea (Li et al., 2017). In 2022, molecular regulatory mechanisms underlying the asexual sporulation of A. cinnamomea induced by nutrient restriction (Li et al., 2022a) and promoted by iron ion (Li et al., 2023) in submerged fermentation were revealed by transcriptomics. In addition, the excellent effect of A. cinnamomea polysaccharides (APSs) was observed on inflammation alleviation and intestinal flora regulation in antibiotic-induced diarrheic mice (Lu et al., 2022a, 2022b).

Polysaccharides are the main product of submerged fermentation of A. cinnamomea. Several studies reported the significantly higher content of polysaccharides and triterpenes in A. cinnamomea than in other similar fungi, such as Ganoderma lucidum and Volvariella volvacea, which indicates the huge development and research value of APSs (Lee et al., 2014). In addition, APSs have different biological activities, such as anticancer (Wang et al., 2015), anti-inflammatory (Meng et al., 2012), antioxidant (Tsai et al., 2007), liver protection (Liu et al., 2023), and nerve protection properties (Han et al., 2020). Studies on APSs usually focused on its activity in vivo (Liu et al., 2010) or the molecular mechanism of its biological activities (Zhang et al., 2018). However, no study has been dedicated to the comprehensive summary of APSs. Thus, this review summarizes the extraction, isolation, composition, structure, and biological activity of APSs and provides a comprehensive and convenient reference for the future research on APSs.

Extraction of APSs

The extraction and characterization of APSs is a difficult but an important process (Figure 1). Since polysaccharide is a polar macromolecular compound that is often soluble in water but insoluble in organic solvents, both hot water extraction (Tsai, et al., 2007) and salt water extraction (Cheng et al., 2009) have become quick and easy to set up common extraction techniques. Additionally, the polysaccharides can be extracted using a diluted alkali technique. However, this method has potential to destroy the structure of APSs and decrease extraction efficiency (Lu et al., 2014a).

Figure 1. Flowchart of steps for the extraction, isolation, and characterization of APSs.

Prior to the extraction of polysaccharides, it is essential to remove the naturally occurring oil-soluble compounds by some efficient techniques, such as supercritical fluid carbon dioxide (99.5% purity) to reduce interference with polysaccharide extraction (Chen et al., 2007). After removing oil-soluble impurities, crude polysaccharides are obtained by water extraction and ethanol precipitation (Li et al., 2022b). Hot-water extraction can extract most of the polysaccharides from the fruiting body and mycelium of A. cinnamomea. Nevertheless, it is difficult to obtain polysaccharides from cell walls by water extraction. Therefore, polysaccharides can be extracted from cell walls of A. cinnamomea mycelia according to a modified method. Briefly, A. cinnamomea mycelia are successively immersed in 5%, 2%, and 1% NaCl solution (w/v) with magnetic stirring for 1 h. Then the mycelia are defatted with chloroform and methanol for 1 h (Wang et al., 2021). Irrespective of the method used to extract the polysaccharide solution, it is necessary to add 70–80% concentrated ethanol, and finally centrifuge and dissolve the precipitation in hot water to obtain crude polysaccharide solution (Zhang et al., 2018). However, low extraction efficiency and high solvent usage in organic solvent extraction ultimately lead to environmental degradation and pose a threat to human health. Thus, finding alternative solvents is urgently required to have a new high--quality and environment friendly extraction technology.

Crude APSs usually contain proteins, pigments, and several small molecular impurities that require further removal. Proteins are primary impurities and can be removed by the Sevag method (Zhang et al., 2018), trichloroacetic acid (Liu et al., 2018), and enzymatic processes (Perera et al., 2017). The pigments in APSs are removed by oxidation (such as 10% H2O2) (Chen et al., 2017), carbon decolorization (Singh et al., 2013), or macroporous resin adsorption (Zou et al., 2015). Subsequently, tiny molecular contaminants are typically eliminated by dialysis. Ion-exchange column chromatography and gel-column chromatography are the methods commonly used for the removal of molecular contaminants (Li et al., 2022b).

In general, tiny molecular contaminants are first purified by anion-exchange column chromatography and further purified by gel column chromatography. Diethylaminoethyl (DEAE) cellulose, DEAE Sephadex, and DEAE Sepharose are the most commonly used anion exchangers, with DEAE-cellulose as the most often used (Shi, 2016). Finally, the APSs are separated by gel--column chromatography, such as Sephadex G-100, based on their size and structure (Li et al., 2022b). Size-exclusion chromatography (SEC) is suitable for separating polysaccharides using aqueous solution as mobile phase and for separating water-soluble samples. It is also possible to separate and purify APS using gel filtration chromatography (GFC), which uses various salt and buffer solution concentrations as eluents. However, GFC is ineffective for mucopolysaccharide separation. Table 1 lists the reported extraction and isolation conditions for APSs.

Table 1. Extraction and isolation conditions for APSs.

Sources Extraction method Isolation and purification method Research results References
Mycelia Extracted with 80°C water at a 1:100 (w/w) ratio for 6 h Separated by SEC All the polysaccharides from six medicinal fungi showed no toxicity to endothelial cells up to a concentration of 250 μg/mL. The high-molecular weight (MV) polysaccharides in the size range of 2693–2876 kDa and middle-molecular weight polysaccharides in the size range of 304–325 kDa presented good antiangiogenic effects Chen et al. (2005)
Mycelia Reflux extracted thrice with 2-L double-distilled water at 90°C with constant stirring at 400 rpm for 2 h Separated by decantation, and 3 mL of sample (APSs extract) was eluted with 0.05-N NaOH (containing 0.02% NaN3) solution on a Sephadex G-100 column (2.5 × 100 cm2) at a flow rate of 0.5 mL/min The distribution of mean molecular mass of fractionated polysaccharides was in the range of 394–940 kDa. The proximate compositions from APSs fraction revealed that all fractions belonged to the category of glycoprotein, having carbohydrate–protein proportion ranging from 0.29 to 10.79 (w/w) Chen et al. (2007)
Mycelia Extracted with deionized water (2 L) at 30°C for 24 h or at 95°C for 6 h Purified by a DEAE-cellulose ion-exchange column PEF-1, PEMC-1, and PEMH-1 are the major water-soluble polysaccharides in the APSs named ACSC. PEMC-1 and PEMH-1 are protein-containing glycan, while PEF-1 is free of any peptide chain Tsai et al. (2007)
Fruiting body Extracted with distilled water (100 mL) at 100°C for 1 h Separated into six fractions by Amicon Ultra-15 5-, 10-, 30-, 50-, and 100-K centrifugal filter devices (Millipore, County Cork, Ireland) Four different molecular weights (<5, 5–30, 30–100, and >100 kDa) of APSs were obtained. The APSs of MV >100 kDa substantially and dose-dependently reduce the development of neovascularization in vitro Yang et al. (2009)
Fruiting body Extracted with boiling water at a ratio of 1:25 (w/v) for approximately 8–12 h Passed through a Sephadex G50 (Amersham Pharmacia Biotech, Piscataway, NJ, USA) gel filtration column and further purified by an anion-exchange column of DEAE-cellulose Percentage of APSs in the lyophilized extracts of fruiting bodies was more than 98%. The APSs can reduce the expressions of inflammatory mediators at the injured site and circulation, especially in the late stage of sepsis Meng et al. (2012)
Mycelia Extracted with cold water Separated by SEC (90 cm H × 1.6 cm D) at a flow rate of 0.4 mL/min The obtained APSs significantly enhance the phagocytosis and bactericidal activity of J774A.1 murine macrophages against Escherichia coli Perera et al. (2017)
Mycelia Extracted with boiled water for 3 h Purified by DEAE-52 cellulose (2.6 × 20 cm2) and Sephadex G-100 column chromatography (1.1 × 100 cm2) The APSs named ACPS-1 with a MV of 22.96 kDa was obtained and presented an outstanding anticancer activity against cervical and skin cancer cells Zhang et al. (2018)
Mycelia 0.1-M sodium acetate (pH 5.5) containing 5-mM cysteine, 100-mg papain, and 5-mM ethylenediaminetetraacetic acid (EDTA) at 60°C for 24 h Purified by GFC The obtained APSs show anticancer effects by inhibiting the EGFR/ERK signaling pathway Lu et al. (2021)
The cell wall of mycelia 5%, 2%, and 1% NaCl solution (w/v) with magnetic stirring for 1 h, defatted with chloroform and methanol for 1 h, and extracted with distilled water in a boiling water bath for 3 h Purified by cross-flow ultra-filtration with molecular weight (MW) cut-off of 100 kDa (Pelli-con®XL, Millipore, USA) Only 3.82% of mushroom cell wall (MCW) drymatter could be extracted by hot water. About half (49.18%) of the MCW was composed of alkali soluble fractions, while mycelia cold alkali-extracted fraction harvested (39.13%) significantly higher than mycelia hot alkali-extracted fraction Wang et al. (2021)

SEC: size-exclusion chromatography; DEAE: diethylaminoethyl; EGFR: epidermal growth factor receptor; ERK: extracellular signal-regulated kinase; GFC: gel filtration chromatography.

Composition and structure of APSs

A. cinnamomea polysaccharides are classified as A. cinnamomea intracellular polysaccharides (AIPSs) and A. cinnamomea extracellular polysaccharides (AEPSs), which are derived from the mycelium/fruiting body of A. cinnamomea and fermentation broth, respectively. Given the complex chemical structure of polysaccharides, extracted polysaccharides are usually hydrolyzed into monosaccharides while detecting their composition by high-performance liquid chromatography (HPLC) (Mendes et al., 2020), thin-layer chromatography (TLC) (Kwamla and Thomas, 2022), gas chromatography (GC) (Andres et al., 2020), and high-performance anion-exchange chromatography (HPAEC-AED) (Brunt et al., 2020). TLC has low sensitivity and poor separation effect; HPAEC-AED is expensive, and the incomplete degradation of polysaccharides by acid hydrolysis will affect the quantitative analysis. Thus, HPLC and GC are widely used due to their fast separation speed, good separation effect, and good reproducibility.

Su et al. (2016) analyzed the polysaccharides from the fruiting body of A. cinnamomea (collection number: KJ-AC-14) by the 1-phenyl-3-methyl-5--pyrazolonepre-column derivatization method and observed that the monosaccharide composition of APSs includes fucose (2.0 ± 0.1 molar%), galactose (2.9 ± 0.2 molar%), glucose (84.0 ± 1.7 molar%), mannose (7.2 ± 1.9 molar%), rhamnose (1.4 ± 0.3 molar%), and xylose (2.4 ± 0.2 molar%). Cheng et al. (2018) analyzed the AIPSs from strain B86 (Taipei Institute of Forestry, Taiwan) in solid-state fermentation by HPAEC and observed that it consists of fucose (3.08 ± 0.05 µmol/g), glucosamine (8.58 ± 0.10 µmol/g), galactose (35.89 ± 0.67 µmol/g), glucose (600.42 ± 7.20 µmol/g), and mannose (20.24 ± 0.73 µmol/g). Zhang et al. (2018) isolated a polysaccharide, named ACPS-1, from the submerged fermentation broth of A. cinnamomea. Then the authors analyzed the structure of ACPS-1 by DEAE-52 Sephadex G-100 column chromatography, Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR), and discovered that it consisted of mannose, glucose, fucose, xypyranose, arabinose, fructose, and rhamnose at a molar ratio of 31.27:1.77:1.44:1.34:1.00, and its backbone consisted of repeating α and →3), its→6), its →2), and backbone glycosidic linkages.

The composition of AIPSs and AEPSs from the ATCC 200183 strain in submerged fermentation at 26°C and 150 r/min for 12 days was analyzed by the phenol–-sulfuric acid method (Lu et al., 2022a, 2022b). Finally, it was observed that the AIPS consisted of galactose (28.52%), glucose (55.31%), mannose (14.34%), and galactosamine (1.83%) whereas the AEPS comprised glucose (84.73%), galactose (7.84%), mannose (5.27%), galacturonic acid (0.76%), and glucuronic acid (1.4%). Table 2 lists the reported monosaccharide compositions of APSs.

Table 2. Monosaccharide compositions of APSs.

Strains Sources Cultivation mode Monosaccharide composition References
Collection number: KJ-AC-14 Fruiting body - Fucose (2.0 ± 0.1%), galactose (2.9 ± 0.2%), glucose (84.0 ± 1.7%), mannose (7.2 ± 1.9%), rhamnose (1.4 ± 0.3%), and xylose (2.4 ± 0.2%) Su et al. (2016)
Taipei Institute of Forestry: B86 Mycelium 0.5-mM potassium sulfate, 24-g/L potato-dextrose-broth (PDB), and 20-g/L glucose for 49 days Fucose (3.08 ± 0.05 µmol/g), glucosamine (8.58 ± 0.10 µmol/g), galactose (35.89 ± 0.67 µmol/g), glucose (600.42 ± 7.20 µmol/g), and mannose (20.24 ± 0.73 µmol/g) Cheng et al. (2018)
- Mycelium Submerged culture for 10 days Mannose, glucose, fucose, xypyranose, arabinose, fructose, and rhamnose at a molar ratio of 31.27:1.77:1.44:1.34:1.00 Zhang et al. (2018)
ATCC 200183 Mycelium Immersed for 12 days at 150 r/min at 26°C Galactose (28.52%), glucose (55.31%), mannose (14.34%), and galactosamine (1.83%) Lu et al. (2022b)
ATCC 200183 Broth Screened with four layers of gauze after immersion for 12 days at 150 r/min at 26°C Glucose (84.73%), galactose (7.84%), mannose (5.27%), galacturonic acid (0.76%), and glucuronic acid (1.4%) Lu et al. (2022a)

Note: “-” not mentioned.

The structure of APSs was detected using FTIR, hydrogen NMR (H-NMR), carbon NMR (C-NMR), and infrared radiation. Su et al. (2016) analyzed the structure of APSs from the fruiting body of A. cinnamomea by FTIR and observed the presence of pyranose. Zhang et al. (2018) analyzed the structure of APSs from the mycelium of A. cinnamomea in submerged fermentation by FTIR and revealed the presence of pyranose. Lu et al. (2021) explored the structure of a APS type (Na10_SPS-F3) using H-NMR and C-NMR, and reported that it is a 3-o-sulfated malonosaccharide, whose pentose 1,4-β-glc is linked to hexose 1,4-α-glc.

Liu et al. (2017) analyzed the structure of an APS type, named ACW0, by one-dimensional (1D) and 2D NMR and observed that it was a type of mannose–galactan whose main chain was α-d-1,6-Gal. Almost one nonreducing terminal α-D-Man and α-L-Fuc in every six α-d-1,6-Gal residues was attached to C-2. In addition, we analyzed the structures of AIPS and AEPS from the ATCC 200183 strain in submerged fermentation by FTIR and revealed that AEPS was a beta-type glucoside with a pyranose ring whereas AIPS possessed (-C≡C-H) and (C-O) functional groups (Lu et al., 2022a, 2022b). Table 3 lists the results of the structural analysis of APSs. Figure 1 presents the steps involved in the extraction, isolation, and characterization of APSs.

Table 3. Structural analysis of APSs.

Strains Sources Method Structure References
Collection number: KJ-AC-14 Fruiting body FTIR Pyranose ring Su et al. (2016)
- Mycelium FTIR and NMR The main chain of ACW0 polysaccharide is 1,6-Gal, and the branch is located at the C-2 site of 1,2,6-Gal Liu et al. (2017)
- Mycelium FTIR Pyranose ring Zhang et al. (2018)
Taipei Institute of Forestry: B86 Mycelium NMR 3-O-sulfomalonyl glucan with eight 1,4-β-Glc moieties connected with ten 1,4-α-Glc moieties Lu et al. (2021)
ATCC 200183 Broth FTIR β-type glucoside Lu et al. (2022a)

Note: “-” not mentioned.

Bioactivity of APSs

The content of APSs in A. cinnamomea is significantly higher than that of similar fungi, such as Ganoderma lucidum and Volvariella volvacea; this indicates that APSs may possess different bioactivities with great research and development values (Hseu et al., 2002; Song and Yen, 2002).

Safety of APS

Chen et al. (2005) evaluated the toxicity of APS toward endothelial cell (EC) viability and found that APS showed no toxicity to endothelial cells up to a concentration of 250 μg/mL. Tsai et al. (2007) found that all cellular viabilities were higher than 90% if Chang liver cells were treated with APS at concentration of up to 200 μg/mL at 37oC for 24 h, indicating that APS was not cytotoxic to Chang liver cells. Wang et al. (2015) found that serum TGF-β quantity in common mouse was 39.59 ± 5.645 ng/mL, compared to the mice fed with A. cinnamomea β-glucan at 32.8 ± 1.879 ng/mL. There was no significant difference between the two groups. It was obvious that daily oral intake of A. cinnamomea β-glucan does not alter serum TGF-β in normal mice. Zhang et al. (2018) assessed the safety of APSs by monitoring the proliferation of normal mouse spleen cells treated with APSs at concentrations ranging from 25 g/mL to 1000 g/mL; the authors observed that APSs showed no effect on cell viability at any concentration. Moreover, in several cases, the APSs increased cell viability. Thus, APSs were safe enough and noncytotoxic toward normal cells.

Anticancer activity of APSs

To date, the impact of different cancers, such as lung, liver, and breast cancers, on the health of patients is increasingly aggravating. With increase in the incidences of different cancers, the drugs used to treat cancers are also improving constantly. Fungal polysaccharides have been identified as pharmacologically active antitumor components (Wang et al., 2015). A. cinnamomea, as a valuable edible fungus, has active compounds with excellent anticancer effects. In vitro and in vivo studies have revealed that the polysaccharides from the fruiting bodies and mycelium of A. cinnamomea have potent anticancer properties (Ho et al., 2008; Liu et al., 2004).

DNA damage is a characteristic of cancer cells, but it can also be a target for treatments that fight the disease. Zhang et al. (2018) mentioned that a new polysaccharide (ACPS-1) isolated from A. cinnamomea mycelium could cause apoptosis and cell cycle arrest in cervical and skin cancer cells by obstructing the DNA repair pathway controlled by topoisomerase I/tyrosine DNA phosphodiesterase I. Fa et al. (2015) used Lewis lung cancer cells, a highly aggressive mouse lung cancer cell line, to investigate the antimetastatic activity of antrodan (a glycoprotein isolated from the mycelium of A. cinnamomea) by direct or indirect immunomodulatory effects. The results showed that direct and indirect immunomodulatory effects had anti-metastasis potential in mouse lung cancer cells. In addition, at the same concentration (50 and 60 µg/mL), antrodan exhibited a stronger indirect immunomodulatory effect on tumor metastasis than direct effect.

Replacement of several functional groups of polysaccharides with sulfate groups could improve their biological activity (Wang et al., 2013, 2018). Lu et al. (2017a) observed that sulfated polysaccharides (SPS) from A. cinnamomea not only inhibited the survival ability of lung cancer cells but also reduced the expression of transforming growth factor β receptor (TGFR) protein and blocked the intracellular signaling pathway regulated by TGFR, which lead to the migration of lung cancer cells. Moreover, the authors were the first to isolate and identify a 1,4-D-galactoman (B86-III) containing 1,6-branched chains from A. cinnamomea. TGFR and its downstream signals, focal adhesion kinase (FAK), and Slug were involved in the development of lung tumors (Lu et al., 2017b). B86-III inhibited the expression and migration of Slug by downregulating the TGFR I protein and inhibiting the phosphorylation of FAK. Finally, the activity of H1975 lung cancer cells was inhibited (Lu et al., 2017b).

Lu et al. (2018) identified an anticancer sulfated β-(1→4)-D-glucan (denoted as AC-SPS-F3) with long β-(1→6)-Glcp branches and a very high sulfate ratio from A. cinnamomea. It inhibited the action of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin. Furthermore, using gel column chromatography, Lu et al. (2021) further isolated SPS and obtained Na10_SPS-F2 and Na10_SPS-F3, which reduced cell viability by eliciting apoptotic responses. Specifically, Na10_SPS-F3 showed anticancer effects by inhibiting the EGFR/extracellular signal-regulated kinase (ERK) signaling pathway. Lu et al. (2023) discovered a sulfated galactoglucan (3-SS) in A. cinnamomea. It could impair the proliferation of H1975 lung cancer cells through EGFR/ERK/Slug signaling.

In addition, the polysaccharides isolated from A. cinnamomea mycelium (AC-PS) significantly inhibited the proliferation of leukemia U937 cells by activating mononuclear cells (MNCs) (Liu et al., 2004). In their recent study, Lin et al. (2023) discovered that SPS from A. cinnamomea with a molecular weight (MW) of 7.9 kDa (dubbed ZnF3 gene) had dual anticancer properties, killing cancer cells while stimulating macrophages. ZnF3 downregulated the expression of transforming growth factor beta receptor (TGFβ) in lung cancer cells. In parallel, ZnF3 activated macrophages via induction of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretion, nitric oxide (NO) production, and phagocytosis. ZnF3 activated the serine/threonine kinase–rapamycin (AKT/mTOR) pathway and induced M1 type macrophage polarization. Cancer cells co-cultured with ZnF3-stimulated macrophages lead to the inhibition of lung cancer cells.

The main disadvantages of natural compounds are their low solubility and bioavailability, and inability to adhere to prescribed treatments. Nanotechnology is one of the new tools used for diagnosis, treatment, and prevention of cancer (Siddiqui et al., 2012). Therefore, nanotechnology offers another mode to improve the bioavailability of naturally active food ingredients. Kong et al. (2013) successfully prepared silica or silica chitosan nanoparticles coated with APSs. The 3-(4,5--dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide experiments showed that APSs coated with silica or silica chitosan nanoparticles inhibited the growth of 10 different human cancer cell lines but were nontoxic to three normal human cell lines. The APSs encapsulated in silica or silica–chitosan nanoparticles showed better stability and increased antitumor activity. Table 4 lists the reported anticancer activity of APSs.

Table 4. Anticancer activity of APSs.

Sources Cancer type Optimum concentration References
Mycelium Leukemic 100 µg/mL Liu et al. (2004)
Mycelium Lung cancer 50 µg/mL for migration inhibition and 60 µg/mL for invasion inhibition Fa et al. (2015)
Mycelium Lung cancer 200 µg/mL Lu et al. (2017a)
Mycelium Lung cancer 200 µg/mL Lu et al. (2017b)
Mycelium Cervical cancer 1,000 µg/mL Zhang et al. (2018)
Skin cancer 800 µg/mL
Mycelium Lung cancer 550 µg/mL Lu et al. (2018)
Mycelium Lung cancer 800 µg/mL Lu et al. (2021)
Mycelium Lung cancer >400 µg/mL Lin et al. (2023)
Mycelium Lung cancer - Lu et al. (2023)

Hepatoprotective activity of APSs

The active compounds from A. cinnamomea are used for the treatment of liver injury, alcoholic liver, nonalcoholic fatty liver disease, and liver cancer with excellent therapeutic effects (Ruan et al., 2022). APSs can considerably protect the liver. Lee et al. (2002) revealed the good anti-hepatitis B virus (HBV) effects of AC-PS. Ho et al. (2008) observed that the AC-PS had a good protective effect against ethanol-induced liver injury. Tang et al. (2019) isolated a galactosylglucan named ACP2 from A. cinnamomea mycelium and evaluated its anti-inflammatory effects on human L02 (liver cell line) cells. The results showed that ACP2 drastically alleviated endotoxin-induced hepatocyte inflammation by decreasing the expressions of cyclooxygenase-2, IL-1b, TNF-α, and IL-6. Han et al. (2006) isolated a neutral polysaccharide, named ACN2a, from A. cinnamomea mycelium and used it (gavaged 0.4 and 0.8 g/kg/day) to treat liver injury in mice induced by Propionibacterium acnes and lipopolysaccharides (LPS). The results showed that ACN2a could considerably suppress rise in serum aspartate aminotransferase and alanine aminotransferase activities produced by P. acnes and LPS in mice and presented a liver protective effect.

In addition, overexpression of reactive oxygen species (ROS) in liver damage leads to inflammation (Matsuzawa et al., 2005). Yang et al. (2022) reported that APSs could activate the NF-E2-related factor 2 signaling pathway (an important antioxidant signaling pathway). APSs could also inhibit ROS expression, increase the gene expression of superoxide dismutase (SOD), and ultimately reduce the expression of inflammatory cytokines. In general, APSs effectively ameliorated liver damage in mice. Moreover, abnormal expression of nucleotide-binding oligomerization domain (NLRP3; leucine-rich repeat and pyrin domain-containing protein 3, a kind of inflammasome) has been found in various models of liver injury (Duffield et al., 2005). Ruan et al. (2022) proved that APSs could activate the autophagy of Kupffer cells and induce the degradation of NLRP3, which reduced inflammatory response, inhibited the release of inflammatory factors, and provided liver protection. Table 5 lists the reported hepatoprotective activity of APSs.

Table 5. Hepatoprotective activity of APSs.

Sources Hepatoprotective type Optimum concentration References
Mycelium HBV 1,000 U/mL Lee et al. (2002)
Mycelium Propionibacterium acnes and LPS-induced hepatic injury 0.8 g/kg/day Han et al. (2006)
Mycelium Alcohol-induced liver injury 500 mg/L Ho et al. (2008)
Mycelium LPS-induced hepatocyte inflammation 100 µg/mL Tang et al. (2019)
Mycelium Inflammatory response in liver injury 15 mg/L Yang et al. (2022)
Mycelium Liver injury caused by abnormal NLRP3 expression 15 mg/kg Ruan et al. (2022)

Anti-inflammatory activity of APSs

Meng et al. (2012) used APSs to treat the sepsis caused by cecal ligation and puncture in mice, and observed that both polysaccharides from the mycelium and fruiting bodies of A. cinnamomea could reduce the expression of inflammatory mediators at the injured site and circulation, especially in the late stage of sepsis. However, the polysaccharides from the fruiting bodies of A. cinnamomea were more effective than those from the mycelia of A. cinnamomea in lowering inflammatory response. In addition, Wu et al. (2007) demonstrated that an alkaline extraction-isoelectric precipitation fraction in APSs (AC-2) could decrease the synthesis of IL-6, IL-10, monocyte chemoattractant protein (MCP)-5 and NO in LPS-stimulated mouse macrophages. This transcriptional downregulation of IL-6, IL-10, and inducible NO synthase (iNOS) genes resulted in the inhibition of IL-6, IL-10, and iNOS. Zheng et al. (2017) obtained an ACHO (AC polysaccharides at 90°C, an oligosaccharide product) from ACP at 90°C by trifluoroacetic acid degradation and observed that ACHO dramatically reduced the inflammatory reactions induced by LPS in vitro and in vivo by suppressing the expression levels of mRNA for several pro-inflammatory cytokines, including IL-6, IL-8, IL-1, TNF-α, and monocyte chemoattractant protein (MCP-1). The underlying molecular mechanism of ACHO’s anti-inflammatory effect promoted O-GlcNAcylation, which in turn prevented the phosphorylation of p38 mitogen-activated protein kinase and protein kinase B (AKT).

Cheng et al. (2016) isolated polysaccharides and SPS from A. cinnamomea mycelium and used the RAW264.7 macrophages induced by LPS to study the effect of polysaccharides and SPS on inflammatory response. The authors observed that SPS significantly inhibited the release of TNF-α and IL-6 more than non-sulfate polysaccharides, with 100% inhibition. This result indicated that the degree of sulfation of APSs could have a role in anti-inflammatory action. Chiu et al. (2013) further purified APSs by Sepharose CL-6B column chromatography and obtained antrodan. Then the authors showed that antrodan was completely harmless to the RAW 264.7 cell line at doses as high as 400 µg/mL and could reduce the inflammatory response induced by LPS in RAW 264.7 cell line. In addition, antrodan significantly reduced NO production at a low dose of 18.75 µg/mL. This finding indicated that several oligosaccharides with less polymerization typically exhibited better solubility in water, compared to polysaccharides (McCranie et al., 2014). Peng et al. (2015) observed that a unique β-glucan (antrodan) from A. cinnamomea mycelium could be used to treat benign prostatic hyperplasia (BPH). Moreover, kidney and testicular apparent weight was completely unaffected by antrodan, which implied that antrodan therapy was safe.

The effects of AIPS and AEPS on inflammation and intestinal flora disturbance induced by lincomycin hydrochloride (LIH) in mice were investigated in the past studies (Lu et al., 2022a, 2022b). Finally, it was observed that AIPS considerably decreased weight loss, restored immunological organ indices, and markedly reduced the levels of pro-inflammatory cytokines TNF-α and IL-6 in mouse serum. Similarly, AEPS could significantly lessen immune organ damage and lower serum levels of inflammatory factors, such as IL-6 and TNF-α. In addition, AIPS and AEPS could regulate and restore the gut microflora structure in mice by significantly reducing the relative abundance of intestinal harmful microorganisms and improving the relative abundance of intestinal beneficial microorganisms.

Lu et al. (2023) discovered a sulfated galactoglucan (3-SS) in A. cinnamomea. The anti-inflammation effects of 3-SS on RAW264.7 macrophage cells, such as IL-6 inhibition, restoration of LPS-induced IκB protein degradation, and inhibiting LPS-induced TGFRII protein degradation, were confirmed to occur via AKT, ERK1/2, and p-38. Table 6 lists the reported anti-inflammatory activity of APSs.

Table 6. Anti-inflammatory activity of APSs.

Sources Inflammatory type Optimum concentration References
Mycelium LPS-induced gene activation in mouse macrophages 200 mg/L Wu et al. (2007)
Fruiting body and mycelium Polymicrobial sepsis 100 mg/kg Meng et al. (2012)
Mycelium LPS-induced inflammation 18.75 µg/mL Chiu et al. (2013)
Mycelium Benign prostate hyperplasia 30 mg/kg Peng et al. (2015)
Mycelium LPS-induced inflammation 500 µg/mL Cheng et al. (2016)
Mycelium LPS-induced inflammation 100 µg/mL Zheng et al. (2017)
Broth LIH-induced inflammation 0.25 g/kg Lu et al. (2022a)
Mycelium LIH-induced inflammation 0.25 g/kg Lu et al. (2022b)
Mycelium Macrophage cells-induced inflammation - Lu et al. (2023)

LIH: lincomycin hydrochloride; LPS: lipopolysaccharides.

Neuroregulatory effects of APSs

The degeneration and necrosis of dopaminergic neurons have been linked to neuroinflammation, which has been demonstrated to persist in Parkinson’s disease (Hirsch et al., 2012). The polysaccharides from several medicinal and edible fungi or plants, such as Astragalus membranaceus and Lentinula edodes, exert good neuroprotective benefits or inhibitory effects on neuroinflammation and present great potential for clinical application (Huang et al., 2017; Iancu et al., 2005). APSs also present good neuroprotective properties and are effectively used in the treatment of Alzheimer’s disease (Wang et al., 2012). Although A. cinnamomea mycelium contained more polysaccharides than the fruiting body, the polysaccharides from its fruiting body are more potent and have a higher inhibitory effect on cytotoxicity (Wang et al., 2012).

As a neurotoxin, 6-hydroxydopamine (6-OHDA) can be selectively absorbed by dopaminergic neurons of monoamine oxidase and transformed into free radicals that harm nerve cells and cause lesions similar to those in Parkinson’s disease (Zhao et al., 2016). Han et al. (2020) reported that APSs enhanced 6-OHDA expression in a rat model of Parkinson’s disease induced by 6-OHDA. However, APSs did not affect the level of serotonin transmitter but mainly improved the survival and regeneration of dopaminergic neurons. APSs also lowered the NLRP3 expression in inflammasome and completely inhibited the expression of NLRP3 inflammatory body and its inflammatory components. In addition, 6-OHDA may activate ROS-NLRP3 to cause death of dopaminergic neurons; however, APSs can suppress this signal and save dopaminergic neurons (Han et al., 2020). Ultimately, APSs improved the neurobehavior, such as mobility and coordination, of mice with Parkinson’s disease (Han et al., 2019).

Antivascular activity of APSs

Angiogenesis, the growth of new blood vessels from those that already exist, largely occurs during human development and reproduction. Vascular abnormalities are caused by the imbalance between the regulation of angiogenesis boosting and inhibiting factors (Cuvillier, 2017). Endothelial cell migration, proliferation, and tube formation are important processes in angiogenesis. Cancer cells produce large amounts of vascular endothelial growth factor (VEGF), basic fibroblast-like growth factor, IL-8, and transforming growth factor-H, all of which promote recruitment and proliferation of endothelial cells (Ferrara, 2000).

In order to study the link between the structure and functioning of polysaccharides on angiogenesis, Chen et al. (2005) analyzed polysaccharides from six medicinal fungi, including A. cinnamomea, Antrodia malicola, Antrodia xantha, Antrodiella liebmannii, Agaricus murrill, and Rigidoporus ulmarius, and observed an association between effect on angiogenesis and molecular size of polysaccharides. In brief, the medium- and high-molecular weight polysaccharides with a respective size range of 304–325 and 2693–2876 kDa exhibited good antiangiogenic effects. In addition, APSs have a very high content of β-D-glucan (Su et al., 2016), which has (1,3)-β-glucans with (1,6)-β-linked side chains (Lee et al., 2002). The β-glucan from A. cinnamomea shows no genotoxicity (Chen et al., 2018). Cheng et al. (2011) isolated a subcomponent of APS named B85PS-I-V, whose main component is 1,6-α-D-mannogalactan (B85PS-III-1) that consists of galactose and caramel. The authors observed that B85PS-III-1 could inhibit the angiogenesis-related processes of endothelial cell migration and tube formation. Cheng et al. (2005) also observed that APSs reduced the production of cyclin D1 by blocking the VEGF receptor signaling pathway, hence reducing angiogenesis.

Yang et al. (2009) also investigated the immunomodulatory effects of APSs on angiogenesis and revealed that the APSs with MW > 100 kDa substantially and dose--dependently reduced the development of neovascularization in vitro. Moreover, the authors revealed that APSs inhibited the angiogenesis actually via decreasing VEGF secretion in tumor cells and activating mononuclear cells (MNCs) to release abundant IL-12 and interferon-γ (IFN-γ). In addition, SPS presented excellent anti-cancer and anti-inflammatory effects (Lin et al., 2019, 2020). Cheng et al. (2018) stated that SPS considerably inhibited the formation of vascular endothelial cell tubes. Liu et al. (2017) isolated a heterogalactan, named ACW0, from A. cinnamomea and discovered that its sulfated derivative, ACW0-Sul, with sulfate substitutions at C-3 and C-4 of 1,2,6-linked galactose, markedly inhibited the tube formation and motility of human microvascular endothelial cells (HMEC-1). Table 7 lists the reported antivascular activity of APSs.

Table 7. Antivascular activity of APSs.

Sources Antivascular type Optimum concentration References
Mycelium VEGF receptor phosphorylation and interaction with VEGF 0.46 µg/mL Cheng et al. (2005)
Mycelium Inhibition of angiogenesis 100 µg/mL Yang et al. (2009)
Mycelium Inhibition of angiogenesis 7.07 µg/mL Cheng et al. (2011)
Mycelium Inhibition of tube formation and motility of HMEC-1 cells dose dependently 3.5 µM Liu et al. (2017)
Mycelium Inhibition of the formation of vascular endothelial cell tubes 160.92 µg/mL Cheng et al. (2018)

Immune regulation activity of APSs

A. cinnamomea polysaccharides play a good role in immune regulation, especially in promoting T cell activation (Liu et al., 2010). The immune system is a crucial defense mechanism that can identify and eliminate foreign bodies, such as viruses and harmful microbes, identify cancerous cells, and keep the body stable (Miao et al., 2015). The immunological aging process is epidemiologically linked to the majority of common aging-related problems (Gress and Deeks, 2009).

Sheu et al. (2009) isolated a polysaccharide, named ACA, from A. cinnamomea mycelium and observed that ACA was a glycoprotein that could activate macrophages through a toll-like receptors/myeloid differentiation primary response protein (TLR2/MyD88)-dependent mechanism, which directly improved macrophage activity. Chen et al. (2008) investigated the effect of APSs on immune functioning by directing cytokine expression and splenic cell immunological modulation in Schistosoma mansoni-infected mice. After 2, 4, and 6 weeks of oral treatment, the mice exhibited high mRNA expression levels of IFN-γ and TNF-α in vivo and increased levels of immunological factors in spleen cells. In addition, the APSs prevented S. mannii infection in BALB/c mouse model. Liu et al. (2018) revealed that APSs enhanced cyclophosphamide (CTX)-induced immunosuppression in BALB/c mice. In brief, 4 weeks of oral APS treatment improved body weight, organ index, T cell performance, and natural killer cell cytotoxicity of mice. The APSs also successfully boosted the overall antioxidant capacity by promoting the activities of SOD, catalase, and glutathione peroxidase in the serum and spleen and by preventing the rise of ROS and malondialdehyde levels.

Asthma is a chronic disease characterized by airway inflammation caused by dysregulation of cytokines secreted by allergen-specific type 2 T-helper cells. Dendritic cells (DCs) act as both initiator of immune response and inducer of T cell tolerance (Banchereau and Steinman, 1998; Pulendran et al., 2001). Liu et al. (2010) studied the immunomodulatory effects of polysaccharides, named GF2, from A. cinnamomea on dendritic cells and its capability to prevent ovalbumin-induced asthma in an allergic asthma mouse model, and observed that GF2 could be utilized as an adjuvant to prevent the development of allergic asthma by developing immunological tolerance. Lin et al. (2015) further studied the effect of high-molecular weight APS (hmwAPS) on immune functioning of dendritic cells and revealed that hmwAPS promoted the production of proinflammatory cytokines and the maturation of dendritic cells. The authors mentioned that hmwAPS presented more activities than low-molecular weight APS. Perera et al. (2018) showed that preconditioning of galactomannan from A. cinnamomea galactomannan (ACGM) enhanced immune response to invading bacteria early in infection but reduced the risk of severe inflammation later by reducing the secretion of pro-inflammatory cytokines. In addition, ACGM showed endotoxin-like effects on mouse macrophages. Table 8 lists the reported immune regulation activity of APSs.

Table 8. Immune regulation activity of APSs.

Sources Immune regulation type Optimum concentration References
Mycelium Cell-mediated immunity - Chen et al. (2008)
Mycelium Cell-mediated immunity 10 µg/mL Sheu et al. (2009)
Mycelium Immunomodulatory effects on dendritic cells 200 µg/mL Liu et al. (2010)
Mycelium Activated dendritic cells 10 µg/mL Lin et al. (2015)
Mycelium CTX-induced immunosuppression 30 mg/kg Liu et al. (2018)
Mycelium Immunomodulatory effects on J774A.1 mouse macrophages, mouse peritoneal macrophages, and human dendritic cells - Perera et al. (2018)

Antioxidant activity of APSs

Oxidation is essential for numerous organisms to generate energy to drive biological processes. Normal energy metabolism in the brain necessitates the consumption of large amounts of oxygen by neurons to maintain biochemical activities in the body (Kawai et al., 1989). According to a growing body of research, oxidative stress-induced cellular damage sets off the physiological processes of aging and most of the pathological developments that eventually result in significant health issues, such as Parkinson’s and Alzheimer’s diseases (Benzi and Moretti, 1995; Finkel and Holbrook, 2000).

Polysaccharides are the main natural antioxidant component of A. cinnamomea. Lin et al. (2010) indicated that in addition to the capability to scavenge 2,2-diphenyl-1--picrylhydrazyl free radicals, APSs had a strong antioxidant activity and thus could be used effectively as components of healthy or functional foods to alleviate oxidative stress response. Song and Yen (2002) showed the significant free radical-scavenging activity of polysaccharides from mycelia of Antrodia cinnamomea (PMAC). Tsai et al. (2007) also revealed that APSs in submerged fermentation exhibited a significant dose-dependent protective effect (of up to 200 µg/mL) on H2O2-induced DNA damage, which could alleviate H2O2-induced oxidative damage.

LPSs reduce the antioxidant and anti-inflammatory capacity, and damage the structure of cecal microflora of yellow-plumed chicken liver (Ye et al., 2022). Ye et al. observed that the addition of APSs to feed improved the health conditions of chickens. This could be due to the combined effects of APSs on antioxidant and cytokine content and restoration of the declined beneficial flora of the cecum. As a result, the APSs improved the quality and yield of yellow feather chicken.

Production of APSs

As the wild fruiting body of A. cinnamomea is scarce and expensive, submerged fermentation has become the most efficient means to produce APSs (Li et al., 2015). APS production can be dramatically improved by optimizing the fermentation conditions (such as temperature and pH) and medium composition (such as carbon, nitrogen, mineral sources, and vitamins) using a hybrid approach of artificial neural network and response surface model (Lin et al., 2006, 2007). Sterol-type activators, including squalene, cholesterol, and stigmasterol, can increase APS production (Lin et al., 2020). Squalene can greatly increase the contents of glucose, fucose, and mannose in APSs. Moreover, the addition of citrus peel extract benefits mycelial growth and AIPS production (Yang et al., 2012). APS yield can also be increased by a two-stage pH fermentation process in shaker culture and stir-tank fermentation (Shu and Lung, 2004). During fermentation, the higher the oxygen supply, the more the APSs produced and the shorter the culture period (Shih et al., 2006). In addition, the microparticle-enhanced cultivation technology can be used to increase the yield of bioactive compounds from A. cinnamomea in submerged fermentation (Fan et al., 2023).

The residue of A. cinnamomea mycelium retains valuable active components after the extraction of polysaccharides or triterpenes. The dietary fiber extracted from the residue of A. cinnamomea mycelium has a high purity and good adsorption capacity to oil, cholesterol, and sodium cholate in vitro (Xia et al., 2022). In addition, using the A. cinnamomea mycelium residue as a feed additive in aquaculture significantly increased zebrafish feed efficiency and decreased fish inflammatory disease symptoms (Chang et al., 2020). Thus, the reuse of A. cinnamomea residue provides a possible opportunity for the recycling economy of the A. cinnamomea industry to maximize utilization of A. cinnamomea and reduce economic losses.

Summary and the future perspectives

This review outlined recent research findings on APSs and summarized their extraction, isolation, composition, structure, and production. In addition, the biological activities, including anticancer, hepatoprotective, anti-inflammatory, neuroregulatory effects, antivascular, immune regulation, and antioxidant activity of APSs, were summarized. However, as a burgeoning resource, the development and application of A. cinnamomea is restricted by the following problems.

First, strengthening the industrialization of APSs requires breaking through the artificial cultivation technology of A. cinnamomea. At present, problems, such as long cultivation cycle, limited or insufficient source of raw materials, and poor fruiting body quality, are the bottlenecks restricting its industrialization development. Therefore, improving approaches, such as exploration of cultivation raw materials and innovation of cultivation methods, can alleviate the slow development of the industry.

Second, numerous products of APSs have been developed, but most of them are health products made from mycelium culture or crude extract of fermentation broth. Thus, the specific contents of the extracts are unclear, which is not conducive to the study and analysis of their pharmacological effects. In the future, further research on the separation and purification of monomers from A. cinnamomea could be carried out, which would lay foundation for the development of A. cinnamomea drugs.

In addition, the pharmacological effectiveness of APSs has been demonstrated at the molecular, cellular, and animal levels. However, a limited number of cases have reported their use in clinical adjuvant therapy or the treatment of major diseases. Therefore, the prospect of APSs in biomedicine can be further expanded, with a focus on the treatment or adjuvant therapy of major diseases.

Finally, throughout the A. cinnamomea research axis, major breakthroughs in germplasm resource protection and innovation, artificial cultivation, new compound mining, gene function, drug research, and development would be made in the future. Then, the standardization and marketization of the A. cinnamomea industry could be promoted globally to play an important role in the treatment and prevention of human diseases.

Author Contributions

Conceptualization: Hua-xiang Li and Lei Yuan. Methodology: Lei Yuan and Wen-yuan Zhou. Writing: original draft preparation, Jia-ning Dai and Bo-ling Liu; review and editing, Hua-xiang Li and Lei Yuan. Supervision: Dan Ji. Project administration: Hua-xiang Li and Dan Ji. Fund acquisition: Hua-xiang Li and Wen-yuan Zhou. All authors had read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant numbers: 32001661 and 32101964), and the Natural Science Foundation of Jiangsu Province, China (Grant number: BK20190890).

Data Availability Statement

Data are contained within the article and available upon request from the corresponding author.

Conflict of Interest

The authors declared no conflict of interest.

REFERENCES

Andres, B.P., Lina M, L.G. and Gonzalo, T.O., 2020. Volatilome study of the feijoa fruit [Acca sellowiana (O. Berg) Burret] with headspace solid phase microextraction and gas chromatography coupled with massspectrometry. Food Chemistry 328: 127109. 10.1016/j.foodchem.2020.127109

Banchereau, J. and Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392: 245–252. 10.1038/32588

Benzi, G. and Moretti, A., 1995. Age-and peroxidative stress--related modifications of the cerebral enzymatic activities linked to mitochondria and the glutathione system. Free Radical Biology and Medicine 19: 77–101. 10.1016/0891-5849(94)00244-E

Brunt, K., Sanders, P., Nota, V.E. and Soest, J.V., 2021. Results multi-laboratory trial ISO/CD 22184-IDF/WD 244: milk and milk products–determination of the sugar contents–high--performance anion exchange chromatography method with pulsed amperometric detection. Journal of AOAC International 104: 732–756. 10.1093/jaocint/qsaa092

Cao, L.P., Zhang, Q., Miao, R.Y., Lin, J.B., Feng, R.C., Ni, Y.Q., Li, W.S., Yang, D.L. and Zhao, X., 2023. Application of omics technology in the research on edible fungi. Current Research in Food Science 6: 100430. 10.1016/j.crfs.2022.100430

Chang, C.C., Lu, Y.C., Wang, C.C., Ko, T.L., Chen, J.R., Wang, W., Chen, Y.L., Wang, Y.W., Chang, T.H. and Hsu, H.F., 2020. Antrodia cinnamomea extraction waste supplementation promotes thermal stress tolerance and tissue regeneration ability of zebrafish. Molecules 25: 4213. 10.3390/molecules25184213

Chen, S.N., Chang, C.S., Chen, S. and Soni, M., 2018. Subchronic toxicity and genotoxicity studies of Antrodia mushroomβ--glucan preparation. Regulatory Toxicology and Pharmacology 92: 429–438. 10.1016/j.yrtph.2017.12.022

Chen, Y.J., Cheng, P.C., Lin, C.N., Liao, H.F., Chen, Y.Y., Chen, C.C. and Li, K.W., 2008. Polysaccharides from Antrodia camphorata mycelia extracts possess immunomodulatory activity and inhibits infection of Schistosoma mansoni. International Immunopharmacology 8: 458–467. 10.1016/j.intimp.2007.11.008

Chen, C.C., Liu, Y.W., Ker, Y.B., Wu, Y.Y., Lai, E.Y., Chyau, C.C., Hseu, T.H. and Peng, R.Y., 2007. Chemical characterization and anti-inflammatory effect of polysaccharides fractionated from submerge-cultured Antrodia camphorata mycelia. Journal of Agricultural and Food Chemistry 55: 5007–5012. 10.1021/jf063484c

Chen, S.C., Lu, M.K., Cheng, J.J. and Wang, D.L., 2005.Antiangiogenic activities of polysaccharides isolated from medicinal fungi. FEMS Microbiology Letters 249: 247–254. 10.1016/j.femsle.2005.06.033

Chen, Q.L., Tang, H.L., Zha, Z.Q., Yin, H.P., Wang, Y., Wang, Y.F., Li, H.T. and Yue, L., 2017. β-D-glucan from Antrodia camphorata ameliorates LPS-induced inflammation and ROS production in human hepatocytes. International Journal of Biological Macromolecules 104: 768777. 10.1016/j.ijbiomac.2017.05.191

Cheng, J.J., Chao, C.H., Chang, P.C. and Lu, M.K., 2016. Studies on anti-inflammatory activity of sulfated polysaccharides from cultivated fungi Antrodia cinnamomea. Food Hydrocolloid 53: 37–45. 10.1016/j.foodhyd.2014.09.035

Cheng, J.J., Chao, C.H. and Lu, M.K., 2018. Large-scale preparation of sulfated polysaccharides with anti-angionenic and anti-inflammatory properties from Antrodia cinnamomia. International Journal of Biological Macromolecules 11: 1198–1205. 10.1016/j.ijbiomac.2018.03.056

Cheng, J.J., Huang, N.K., Chang, T.T., Wang, D.L. and Lu, M.K., 2005. Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Science. 76: 3029–3042. 10.1016/j.lfs.2004.11.023

Cheng, J.J., Huang, N.K., Lur, H.S., Kuo, C.I. and Lu, M.K., 2009. Characterization and biological functions of sulfated polysaccharides from sulfated-salt treatment of Antrodia cinnamomea. Process Biochemistry 44: 453–459. 10.1016/j.procbio.2008.12.012

Chen, S.C., Lu, M.K., Cheng, J.J. and Wang, D.L., 2005. Antiangiogenic activities of polysaccharides isolated from medicinal fungi. FEMS Microbiology Letters 249: 247–254. 10.1016/j.femsle.2005.06.033

Cheng, J.J., Lu, M.K., Lin, C.Y. and Chang, C.C., 2011. Characterization and functional elucidation of a fucosylated 1,6-α-D-mannogalactan polysaccharide from Antrodia cinnamomea. Carbohydrate Polymers 83: 545–553. 10.1016/j.carbpol.2010.08.016

Chiu, C.H., Peng, C.C., Ker, Y.B., Chen, C.C., Lee, A., Chang, W.L., Chyau, C.C. and Peng, R.Y., 2013. Physicochemical characteristics and anti-inflammatory activities of antrodan, a novel glycoprotein isolated from Antrodia cinnamomea mycelia. Molecules 19: 22–40. 10.3390/molecules19010022

Cuvillier, O., 2017. The therapeutic potential of HIF-2 antagonism in renal cell carcinoma. Translational Andrology and Urology 6: 131–133. 10.21037/tau.2017.01.12

Duffield, J.S., Forbes, S.J., Constandinou, C.M., Clay, S., Partolina, M. and Vuthoori, S., 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. Journal of Clinical Investigation 115: 56–65. 10.1172/JCI200522675

Fa, K.N., Yang, C.M., Chen, P.C., Lee, Y.Y., Chyau, C.C. and Hu, M.L., 2015. Anti-metastatic effects of antrodan, the Antrodia cinnamomea mycelia glycoprotein, in lung carcinoma cells. International Journal of Biological Macromolecules 74: 476–482. 10.1016/j.ijbiomac.2015.01.004

Fan, J.H., Lai, K.S., Huang, Y.Y., Chen, H.Y., Xiong, L.Q., Guo, H.K., Yang, Q.Q. and Zhang, B.B., 2023. Efficient production of Antrodin C by microparticle-enhanced cultivation of medicinal mushroom Antrodia cinnamomea. Journal of Bioscience and Bioengineering 135: 232–237. 10.1016/j.jbiosc.2022.12.013

Ferrara, N., 2000. VEGF: an update on biological and therapeutic aspects. Current Opinion in Biotechnology 11: 617–624. 10.1016/S0958-1669(00)00153-1

Finkel, T. and Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408: 239–247. 10.1038/35041687

Geng, Y.Y., Zhang, S.X., Yang, N.X. and Qin, L.K., 2022. Whole-genome sequencing and comparative genomics analysis of the wild edible mushroom (Gomphus purpuraceus) provide insights into its potential food application and artificial domestication. Genes 13: 1628. 10.3390/genes13091628

Gress, R.E. and Deeks, S.G., 2009. Reduced thymus activity and infection prematurely age the immune system. Journal of Clinical Investigation 119: 2884–2887. 10.1172/JCI40855

Han, C.Y., Guo, L., Yang, Y., Li, W.Y., Sheng, Y.J., Wang, J., Guan, Q.B. and Zhang, X.L., 2019. Study on Antrodia camphorata polysaccharide in alleviating the neuroethology of PD mice by decreasing the expression of NLRP3 inflammasome. Phytotherapy Research 33: 2288–2297. 10.1002/ptr.6388

Han, H.F., Nakamura, N., Zuo, F., Hirakawa, A., Yokozawa, T. and Hattori, M., 2006. Protective effects of a neutral polysaccharide isolated from the mycelium of Antrodia cinnamomea on Propionibacterium acnes and lipopolysaccharide-induced hepatic injury in mice. Chemical and Pharmaceutical Bulletin 54: 496–500. 10.1248/cpb.54.496

Han, C.Y., Shen, H.P., Yang, Y., Sheng, Y.J., Wang, J., Li, W.Y., Zhou, X.H., Guo, L., Zhai, L.P. and Guan, Q.,B 2020. Antrodia camphorata polysaccharide resists 6-OHDA-induced dopaminergic neuronal damage by inhibiting ROS-NLRP3 activation. Brain Behavour 10: e01824. 10.1002/brb3.1824

He, R.J., Wu, K.X., Zhang, A.Q., Xie, Z.F. and Sun, P.L., 2019. Mechanochemical-assisted extraction and pharmacological study of triterpenoids from Antrodia camphorata. Applied Sciences 9: 4281. 10.3390/app9204281

Hirsch, E.C., Vyas, S. and Hunot, S., 2012. Neuroinflammation in Parkinson’s disease. Parkinsonism & Related Disorders 18: S210–S212. 10.1016/S1353-8020(11)70065-7

Ho, Y.C., Lin, M.T., Duan, K.J. and Chen, Y.S., 2008. The hepatoprotective activity against ethanol-induced cytotoxicity by aqueous extract of Antrodia cinnamomea. Journal of the Chinese Institute of Chemical Engineers 39: 441–447. 10.1016/j.jcice.2008.03.008

Hseu, Y.C., Chang, W.C., Hseu, Y.T., Lee, C.Y., Yech, Y.J., Chen, P.C., Chen, J.Y. and Yang, H.L., 2002. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sciences 71: 469–482. 10.1016/S0024-3205(02)01686-7

Hsieh, T.C., Wu, P., Park, S. and Wu, J.M., 2006. Induction of cell cycle changes and modulation of apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression by water extracts of I’m-Yunity (PSP). BMC Complementary Medicine and Therapies 6: 30. 10.1186/1472-6882-6-30

Huang, Y.C., Tsay, H.J., Lu, M.K., Lin, C.H., Yeh, C.W., Liu, H.K. and Shiao, Y.J., 2017. Astragalus membranaceus polysaccharides ameliorates obesity, hepatic steatosis, neuroinflammation and cognition impairment without affecting amyloid deposition in metabolically stressed APPswe/PS1dE9 mice. International Journal of Molecular Sciences 18: 2746. 10.3390/ijms18122746

Iancu, R., Mohapel, P., Brundin, P. and Paul, G., 2005. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson’s disease in mice. Behavioural Brain Research 162: 1–10. 10.1016/j.bbr.2005.02.023

Kawai, S., Yonetani, M., Nakamura, H. and Okada, Y., 1989. Effects of deprivation of oxygen and glucose on the neural activity and the level of high energy phosphates in the hippocampal slices of immature and adult rat. Developmental Brain Research 48: 11–18. 10.1016/0165-3806(89)90089-8

Kong, Z.L., Chang, J.S. and Chang, K.L.B., 2013. Antiproliferative effect of Antrodia camphorata polysaccharides encapsulated in chitosan–silica nanoparticles strongly depends on the metabolic activity type of the cell line. Journal of Nanoparticle Research 15: 1945. 10.1007/s11051-013-1945-2

Kwamla, A.R. and Thomas, S., 2022. Detection of mycolactone by thin layer chromatography. Methods in Molecular Biology 2387: 131–149. 10.1007/978-1-0716-1779-3_14

Lavi, I., Friesem, D., Geresh, S., Hadar, Y. and Schwartz, B., 2006. An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells. Cancer Letters 244: 61–70. 10.1016/j.canlet.2005.12.007

Lee, T.H., Chen, C.C., Chen, J.J., Liao, H.F., Chang, H.S., Sung, P.J., Tseng, M.H., Wang, S.Y., Ko, H.H. and Kuo, Y.H., 2014. New and cytotoxic components from Antrodia camphorata. Molecules 19: 21378–21385. 10.3390/molecules191221378

Lee, I.H., Huang, R.L., Chen, C.T., Chen, H.C., Hsu, W.C. and Lu, M.K., 2002.Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiology Letters 209: 63-67.10.1111/j.1574-6968.2002.tb11110.x

Lee, J.C., Lee, K.Y., Son, Y.O., Choi, K.C., Kim, J., Truong, T.T. and Jang, Y.S., 2005. Plant-originated glycoprotein, G-120, inhibits the growth of MCF-7 cells and induces their apoptosis. Food and Chemical Toxicology 43: 961-968.10.1016/j.fct.2005.02.002

Li, H.X., Dai, J.N., Shi, Y., Zhu, X.Y., Jia, L.Q. and Yang, Z.Q., 2023. Molecular regulatory mechanism of the iron-ion-promoted asexual sporulation of Antrodia cinnamomea in submerged fermentation revealed by comparative transcriptomics. Journal of Fungi 9: 235. 10.3390/jof9020235

Li, H.X., Ji, D., Luo, Z.S., Ren, Y.L., Lu, Z.M., Yang, Z.Q. and Xu. Z.H., 2022a. Comparative transcriptomic analyses reveal the regulatory mechanism of nutrient limitation-induced sporulation of Antrodia cinnamomea in submerged fermentation. Foods 11: 2715. 10.3390/foods11172715

Li, H.X., Lu, Z.M., Geng, Y., Gong, J.S., Zhang, X.J., Shi, J.S., Xu, Z.H. and Ma, Y.H., 2015. Efficient production of bioactive metabolites from Antrodia camphorata ATCC 200183 by asexual reproduction-based repeated batch fermentation. Bioresource Technology 194: 334–343. 10.1016/j.biortech.2015.06.144

Li, H.X., Lu, Z.M., Zhu, Q., Gong, J.S., Geng, Y., Shi, J.S., Xu, Z.H. and Ma, Y.H., 2017. Comparative transcriptomic and proteomic analyses reveal a fluG-mediated signaling pathway relating to asexual sporulation of Antrodia camphorata. Proteomics 17: 1700256. 10.1002/pmic.201700256

Li, H.X., Wang, J.J., Lu, C.L., Gao, Y.J., Gao, L. and Yang, Z.Q., 2022b. Review of bioactivity, isolation, and identification of active compounds from Antrodia cinnamomea. Bioengineering (Basel) 9: 494. 10.3390/bioengineering9100494

Lin, E.S. and Chen, Y.H., 2007. Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media. Bioresource Technology 98: 2511–2517. 10.1016/j.biortech.2006.09.008

Lin, Z.H., Lu, M.K., Lo, H.C., Chang, C.C., Tseng, A.J., Chao, C.H. and Lin, T.Y., 2023. ZnF3, a sulfated polysaccharide from Antrodia cinnamomea inhibits lung cancer cells via induction of apoptosis and activation of M1-like macrophage-induced cell death. International Journal of Biological Macromolecules 238: 124144. 10.1016/j.ijbiomac.2023.124144

Lin, T.Y., Lu, M.K., Tseng, A.J. and Chao, C.H., 2020. Effects of sterol-type elicitors on biochemical characterization of polysaccharides from Antrodia cinnamomea. International Journal of Biological Macromolecules 162: 1476–1483. 10.1016/j.ijbiomac.2020.07.201

Lin, C.C., Pan, I.H., Li, Y.R., Pan, Y.G., Lin, M.K., Lu, Y.H., Wu, H.C. and Chu, C.L., 2015. The adjuvant effects of high--molecule-weight polysaccharides purified from Antrodia cinnamomea on dendritic cell function and DNA vaccines. Plos One 10: e0116191. 10.1371/journal.pone.0116191

Lin, E.S. and Sung, S.C., 2006. Cultivating conditions influence exopolysaccharide production by the edible Basidiomycete Antrodia cinnamomea in submerged culture. International Journal of Food Microbiology 108: 182–187. 10.1016/j.ijfoodmicro.2005.11.010

Lin, T.Y., Tseng, A.J., Qiu, W.L., Chao, C.H. and Lu, M.K., 2019. A sulfated glucan from Antrodia cinnamomea reduces slug expression through regulation of TGFbeta/AKT/GSK3beta axis in lung cancer. Carbohydrate Polymers 210: 175–184. 10.1016/j.carbpol.2019.01.078

Lin, E.S., Yang, C.T., Chou, H.J. and Chang, T.T., 2010. Screening of antioxidant activities by the edible basidiomycete Antrodia Cinnamomea strains in submerged culture. Journal of Food Biochemistry 34: 1141–1156. 10.1111/j.1745-4514.2010.00355.x

Liu, Y.Q., Ding, Y.Q., Ye, M., Zhu, T., Tian, D.B. and Ding, K., 2017. A novel heterogalactan from Antrodia camphorata and anti-angiogenic activity of its sulfated derivative. Polymers 9: 228. 10.3390/polym9060228

Liu, J.J., Huang, T.S., Hsu, M.L., Chen, C.C., Lin, W.S., Lu, F.J. and Chang, W.H., 2004. Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicol. Appl. Pharm. 201: 186–193. 10.1016/j.taap.2004.05.016

Liu, K.J., Leu, S.J., Su, C.H., Chiang, B.L., Chen, Y.L. and Lee, Y.L., 2010. Administration of polysaccharides from Antrodia camphorata modulates dendritic cell function and alleviates allergen-induced T helper type 2 responses in a mouse model of asthma. Immunology 129: 351–362. 10.1111/j.1365-2567.2009.03175.x

Liu, F., Ooi, V.E. and Chang, S.T., 1997. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sciences 60: 763–771. 10.1016/s0024-3205(97)00004-0

Liu, Y.G., Yang, A.H., Qu, Y.D., Wang, Z.Q., Zhang, Y.Q., Liu, Y., Wang, N., Teng, L.R. and Wang, D., 2018. Ameliorative effects of Antrodia cinnamomea polysaccharides against cyclophosphamide-induced immunosuppression related to Nrf2/HO-1 signaling in BALB/c mice. International Journal of Biological Macromolecules 116: 8–15. 10.1016/j.ijbiomac.2018.04.178

Liu, X.F., Yu, S.Z., Zhang, Y., Zhang, W., Zhong, H., Lu, X.Q. and Guan, R.F., 2023. A review on the protective effect of active components in Antrodia camphorata against alcoholic liver injury. Journal of Ethnopharmacology 300: 115740. 10.1016/j.jep.2022.115740

Lu, M.K., Chao, C.H., Chang, T.Y., Cheng, M.C., Hsu, Y.C. and Chang, C.C., 2023. A branched 2-O sulfated 1,3-/1,4-galactoglucan from Antrodia cinnamomea exhibits moderate antiproliferative and anti-inflammatory activities. International Journal of Biological Macromolecules 241: 124559. 10.1016/j.ijbiomac.2023.124559

Lu, M.K., Chao, C.H., Hsu, Y.C. and Chang, C.C., 2021. Structural sequencing and anti-inflammatory, anti-lung cancer activities of 1,4-α/β-sulfomalonoglucan in Antrodia cinnamomea. International Journal of Biological Macromolecules 170: 307–316. 10.1016/j.ijbiomac.2020.12.135

Lu, M.J., Fan, W., Wang, W., Chen, T.C., Tang, Y.C., Chu, F.H., Chang, T.T., Wang, S.Y., Meng, Y., Chen, Y.H., Lin, Z.S., Yang, K.J., Chen, S.M., Teng, Y.C., Lin, Y.L., Shaw, J.F., Wang, T.F. and Li, W.H., 2014a. Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development. Proceedings of the National Academy of Sciences 44: E4743–E4752. 10.1073/pnas.1417570111

Lu, Z.M., He, Z., Li, H.X., Gong, J.S., Geng, Y., Xu, H.Y., Shi, J.S. and Xu, Z.H., 2014b. Modified arthroconidial inoculation method for the efficient fermentation of Antrodia camphorata ATCC 200183. Biochemical Engineering Journal 87: 41–49. 10.1016/j.bej.2014.03.020

Lu, M.K., Lee, M.H., Chao, C.H. and Hsu, Y.C., 2020. Physiochemical changes and mechanisms of anti-inflammation effect of sulfated polysaccharides from ammonium sulfate feeding of Antrodia cinnamomea. International Journal of Biological Macromolecules 148: 715–721. 10.1016/j.ijbiomac.2020.01.110

Lu, C.L., Lee, B.H., Ren, Y.L., Ji, D., Rao, S.Q. and Li, H.X., 2022a. Effects of exopolysaccharides from Antrodia cinnamomea on inflammation and intestinal microbiota disturbance induced by antibiotics in mice. Food Biosciences 50: 102116. 10.1016/j.fbio.2022.102116

Lu, Z.M., Lei, J.Y., Xu, H.Y., Shi, J.S. and Xu, Z.H., 2011. Optimization of fermentation medium for triterpenoid production from Antrodia camphorata ATCC 200183 using artificial intelligence-based techniques. Applied Microbiology and Biotechnology 92: 371–379. 10.1007/s00253-011-3544-4

Lu, C.L., Li, H.X., Zhu, X.Y., Luo, Z.S., Rao, S.Q. and Yang, Z.Q., 2022b. Regulatory effect of intracellular polysaccharides from Antrodia cinnamomea on the intestinal microbiota of mice with antibiotic-associated diarrhea. Quality Assurance and Safety of Crops & Foods 14: 124–134. 10.15586/qas.v14i3.1073

Lu, M.K., Lin, T.Y. and Chang, C.C., 2018. Chemical identification of a sulfated glucan from Antrodia cinnamomea and its anti-cancer functions via inhibition of EGFR and mTOR activity. Carbohydrate Polymers 202: 536–544. 10.1016/j.carbpol.2018.09.009

Lu, M.K., Lin, T.Y., Chao, C.H., Hu, C.H. and Hsu, H.Y., 2017a. Molecular mechanism of Antrodia cinnamomea sulfated polysaccharide on the suppression of lung cancer cell growth and migration via induction of transforming growth factor β receptor degradation. International Journal of Biological Macromolecules 95: 1144–1152. 10.1016/j.ijbiomac.2016.11.004

Lu, M.K., Lin, T.Y., Hu, C.H., Chao, C.H., Chang, C.C. and Hsu, H.Y., 2017b. Characterization of a sulfated galactoglucan from Antrodia cinnamomea and its anticancer mechanism via TGFbeta/FAK/Slug axis suppression. Carbohydrate Polymers 167: 29–39. 10.1016/j.carbpol.2019.01.078

Matsuzawa, A., Saegusa, K., Noguchi, T., Sadamitsu, C., Nishitoh, H. and Nagai, S., 2005. ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nature Immunology 6: 587–592. 10.1038/ni1200

McCranie, E.K. and Bachmann, B.O., 2014. Bioactive oligosaccharide natural products. Natural Product Reports 31: 1026–1042. 10.1039/c3np70128j

Mendes, N.S., Pereira, S.M.F., Arantes, M.B.S., Glória, L.L., Nunes, C.R., Passos, M.D., Vieira, I.J.C., Rodrigues, R. and Oliveira, D.B., 2020. Bioanalytical method validation for the quantification of the chlorogenic acid in Capsicum baccatum through high performance liquid chromatography (HPLC-DAD). Food Chemistry 325: 126929. 10.1016/j.foodchem.2020.126929

Meng, L.M., Pai, M.H., Liu, J.J. and Yeh, S.L., 2012. Polysaccharides from extracts of Antrodia camphorata mycelia and fruiting bodies modulate inflammatory mediator expression in mice with polymicrobial sepsis. Nutrition 28: 942–949. 10.1016/j.nut.2012.01.006

Miao, M.S., Cheng, B.L., Guo, L. and Shi, J.J., 2015. Effects of Fuzheng Paidu tablet on peripheral blood T lymphocytes, intestinal mucosa T lymphocytes, and immune organs in cyclophosphamide-induced immunosuppressed mice. Human Vaccines & Immunotherapeutics 11: 2659–2663. 10.1080/21645515.2015.1082694

Peng, C.C., Lin, Y.T., Chen, K.C., Chyau, C.C. and Peng, R.Y., 2015. Antrodan, a β-glucan obtained from Antrodia cinnamomea mycelia, is beneficial to benign prostate hyperplasia. Food & Function 6: 635–645. 10.1039/c4fo00472h

Perera, N., Yang, F.L., Chang, C.M., Lu, Y.T., Zhan, S.H., Tsai, Y.T., Hsieh, J.F., Li, L.H., Hua, K.F. and Wu, S.H., 2017. Galactomannan from Antrodia cinnamomeaenhances the phagocytic activity of macrophages. Organic Letters 19: 3486–3489. 10.1021/acs.orglett.7b01468

Perera, N., Yang, F.L., Lu, Y.T., Li, L.H., Hua, K.F. and Wu, S.H., 2018. Antrodia cinnamomea galactomannan elicits immuno-stimulatory activity through toll-like receptor 4. International Journal of Biology Sciences 14: 1378–1388. 10.7150/ijbs.24564

Pulendran, B., Banchereau, J., Maraskovsky, E. and Maliszewski, C., 2001. Modulating the immune response with dendritic cells and their growth factors. Trends in Immunology 22: 41–47. 10.1016/S1471-4906(00)01794-4

Ruan, S.L., Yang, Y. and Li, W.Y., 2022. Antrodia Camphorata polysaccharide activates autophagy and regulates NLRP3 degradation to improve liver injury-related inflammatory response. Aging 14: 8970–8981. 10.18632/aging.204330

Sheu, F., Chien, P.J., Hsieh, K.Y., Chin, K.L., Huang, W.T., Tsao, C.Y., Chen, Y.F., Cheng, H.C. and Chang, H.H., 2009. Purification, cloning, and functional characterization of a novel immunomodulatory protein from Antrodia camphorata (bitter mushroom) that exhibits TLR2-dependent NF-kappaB activation and M1 polarization within murine macrophages. Journal of Agricultural and Food Chemistry 57: 4130–4141. 10.1021/jf900469a

Shi, L., 2016. Bioactivities, isolation and purification methods of polysaccharides from natural products: a review. International Journal of Biological Macromolecules 92: 37–38. 10.1016/j.ijbiomac.2016.06.100

Shih, I.L., Pan, K. and Hsieh, C.Y., 2006. Influence of nutritional components and oxygen supply on the mycelial growth and bioactive metabolites production in submerged culture of Antrodia cinnamomea. Process Biochemistry 41: 1129–1135. 10.1016/j.procbio.2005.12.005

Shu, C.H. and Lung, M.Y., 2004. Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures. Process Biochemistry 39: 931–937. 10.1016/S0032-9592(03)00220-6

Siddiqui, I.A., Adhami, V.M., Chamcheu, J.C. and Mukhtar, H., 2012. Impact of nanotechnology in cancer: emphasis on nanochemoprevention. International Journal of Nanomedicine 7: 591–605. 10.2147/IJN.S26026

Singh, K., Bharose, R., Verma, S.K. and Singh, V.K., 2013. Potential of powdered activated mustard cake for decolorising raw sugar. Journal of the Science of Food and Agriculture 93: 157–165. 10.1002/jsfa.5744

Song, T.Y. and Yen, G.C., 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J. Agr. Food. Chem. 50: 3322–3327. 10.1021/jf011671z

Su, C.H., Hsieh, Y.C., Chng, J.Y., Lai, M.N. and Ng, L.T., 2023. Metabolomic profiling of different Antrodia cinnamomea phenotypes. Journal of Fungi 9: 97. 10.3390/jof9010097

Su, C.H., Lai, M.N., Lin, C.C. and Ng, L.T., 2016. Comparative characterization of physicochemical properties and bioactivities of polysaccharides from selected medicinal mushrooms. Applied Microbiology and Biotechnology 100: 4385–4393. 10.1007/s00253-015-7260-3

Tang, H.L., Nie, W.B., Xiao, J.N., Zha, Z.Q., Chen, Q.L. and Yin, H.P., 2019. Structural characterization and anti-inflammatory effect in hepatocytes of a galactoglucan from Antrodia camphorata mycelium. RSC Advances 9: 7664–7672. 10.1039/c8ra10347j

Tsai, M.C., Song, T.Y., Shih, P.H. and Yen, G.C., 2007. Antioxidant properties of water-soluble polysaccharides from Antrodia cinnamomea in submerged culture. Food Chemistry 104: 1115–1122. 10.1016/j.foodchem.2007.01.018

Wang, J.L., Bao, A.J., Meng, X.H., Guo, H.Y., Zhang, Y.D. and Zhao, Y.L., 2018. An efficient approach to prepare sulfated polysaccharide and evaluation of anti-tumor activities in vitro. Carbohydrate Polymers 184: 366–375. 10.1016/j.carbpol.2017.12.065

Wang, L.C., Wang, S.E., Wang, J.J., Tsai, T.Y., Lin, C.H., Pan, T.M. and Lee, C.L., 2012. In vitro and in vivo comparisons of the effects of the fruiting body and mycelium of Antrodia camphorata against amyloid β-protein-induced neurotoxicity and memory impairment. Applied Microbiology and Biotechnology 94: 1505–1519. 10.1007/s00253-012-3941-3

Wang, W.J., Wu, Y.S., Chen, S., Liu, C.F. and Chen, S.N., 2015. Mushroom β-glucan may immunomodulate the tumor--associated macrophages in the lewis lung carcinoma. BioMed Research International 2015: 604385. 10.1155/2015/604385

Wang, X.M., Zhang, Z.S., Yao, Q., Zhao, M.X. and Qi, H.M., 2013. Phosphorylation of low-molecular-weight polysaccharide from Enteromorpha linza with antioxidant activity. Carbohydrate Polymers 96: 371–375. 10.1016/j.carbpol.2013.04.029

Wang, Z.Q., Zhu, C.X., Dai, A.R., Chen, L., You, C.P. and Zhang, B.B., 2021. Chemical characterization and antioxidant properties of cell wall polysaccharides from Antrodia cinnamomea mycelia. Food Bioscience 41: 100932. 10.1016/j.fbio.2021.100932

Wu, Y.Y., Chen, C.C., Chyau, C.C., Chung, S.Y. and Liu, Y.W., 2007. Modulation of inflammation-related genes of polysaccharides fractionated from mycelia of medicinal basidiomycete Antrodia camphorata. Acta Pharmacologica Sinica 28: 258–267. 10.1111/j.1745-7254.2007.00500.x

Xia, Y.J., Meng, P., Liu, S.D., Tan, Z.M., Yang, X. and Liang, L.H., 2022. Structural and potential functional properties of alkali-extracted dietary fiber from Antrodia camphorata. Frontiers In Microbiology 13: 921164. 10.3389/fmicb.2022.921164

Yang, Y., Han, C.Y., Sheng, Y.J., Wang, J., Li, W.Y., Zhou, X.H. and Ruan, S.L., 2022. Antrodia camphorata polysaccharide improves inflammatory response in liver injury via the ROS/TLR4/NF-kappaB signal. Journal of Cellular and Molecular Medicine 26: 2706–2716. 10.1111/jcmm.17283

Yang, F.C., Ma, T.W. and Chuang, Y.T., 2012. Medium modification to enhance the formation of bioactive metabolites in shake flask cultures of Antrodia cinnamomea by adding citrus peel extract. Bioprocess and Biosystems Engineering 35: 1251–1258. 10.1007/s00449-012-0712-6

Yang, B., Xiao, B. and Sun, T., 2013. Antitumor and immunomodulatory activity of Astragalus membranaceus polysaccharides in H22 tumor-bearing mice. International Journal of Biological Macromolecules 62: 287–290. 10.1016/j.ijbiomac.2013.09.016

Yang, C.M., Zhou, Y.J., Wang, R.J. and Hu, M.L., 2009. Anti-angiogenic effects and mechanisms of polysaccharides from Antrodia cinnamomea with different molecular weights. Journal of Ethnopharmacology 123: 407–412. 10.1016/j.jep.2009.03.034

Ye, J.L., Zhang, C., Fan, Q.L., Lin, X.J., Wang, Y.B. and Azzam, M., 2022. Antrodia cinnamomea polysaccharide improves liver antioxidant, anti-inflammatory capacity, and cecal flora structure of slow-growing broiler breeds challenged with lipopolysaccharide. Frontiers in Veterinary Science 9: 994782. 10.3389/fvets.2022.994782

Yukawa, H., Ishikawa, S., Kawanishi, T., Tamesada, M. and Tomi, H., 2012. Direct cytotoxicity of Lentinula edodes mycelia extract on human hepatocellular carcinoma cell line. Biological and Pharmaceutical Bulletin 35: 1014–1021. 10.1248/bpb.b110657

Zhang, B.B., Guan, Y.Y., Hu, P.F., Chen, L., Xu, G.R., Liu, L. and Cheng, P.C.K., 2019. Production of bioactive metabolites by submerged fermentation of the medicinal mushroom Antrodia cinnamomea: recent advances and future development. Critical Reviews In Biotechnology 39: 541–554. 10.1080/07388551.2019.1577798

Zhang, Y.T., Wang, Z., Li, D.Y., Zang, W.T., Zhu, H., Wu, P.Y., Mei, Y.X. and Liang, Y.X., 2018. A polysaccharide from Antrodia cinnamomea mycelia exerts antitumor activity through blocking of TOP1/TDP1-mediated DNA repair pathway. International Journal of Biological Macromolecules 120: 1551–1560. 10.1016/j.ijbiomac.2018.09.162

Zhao, J., Cheng, Y.Y., Yang, C.B., Lau, S., Lao, L.X., Shuai, B., Cai, J. and Rong, J.H., 2016. Botanical drug puerarin attenuates 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via upregulating mitochondrial enzyme arginase-2. Molecular Neurobiology 53: 2200–2211. 10.1007/s12035-015-9195-1

Zheng, J.P., Jiao, S.M., Li, Q.Y., Jia, P.Y., Yin, H. and Zhao, X.M., 2017. Antrodia cinnamomea oligosaccharides suppress lipopolysaccharide-induced inflammation through promoting O-GlcNAcylation and repressing p38/Akt phosphorylation. Molecules 23: 51. 10.3390/molecules 23010051

Zou, S.P., Liu, M., Wang, Q.L., Xiong, Y., Niu, K., Zheng, Y.G. and Shen, Y.C., 2014. Preparative separation of echinocandin B from Aspergillus nidulans broth using macroporous resin adsorption chromatography. Journal of Chromatography B. 978: 111–117. 10.1016/j.jchromb.2014.11.028