Sodium sulfate addition increases the bioresource of biologically active sulfated polysaccharides from Antrodia cinnamomea.

Fungal sulfated polysaccharides (SPS) have been used in the pharmaceutical industry. In this study, sodium sulfate was employed as an elicitor to induce stress on the mycelia of Antrodia cinnamomea for the biosynthesis of SPS with high sulfate content. Sodium sulfate treatments increased the yield of SPS to 4.46 % and increased the sulfate content to 6.8 mmol/g of SPS. SPS were extracted from A. cinnamomea cultured with 500 mM sodium sulfate; these SPSs are denoted as Na500. Na500 exhibited the highest sulfate content and dose-dependent inhibitory activity against LPS-induced production of macrophage interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1ß (IL-1ß). Mechanistically, Na500 hindered the phosphorylation of transforming growth factor-ß receptor II (TGFRII), extracellular signal-regulated kinases (ERK), and protein kinase B (AKT) expression. A purified 7.79 kDa galactoglucan, Na500 F3, augmented the anti-inflammation activity by inhibiting LPS-induced TGFß release. Additionally, Na500 F3 restrained the LPS-induced phosphorylation of p-38, ERK, AKT, and TGFRII in RAW264.7 cells. Na500 F3 impeded the proliferation of lung cancer H1975 cells by inhibiting the phosphorylation of focal adhesion kinase, ERK, and Slug. The anti-inflammation and anticancer properties of Antrodia SPS contribute to its health benefits, suggesting its utility in functional foods.

Characterization and Crystal Structures of a Cubebol-Producing Sesquiterpene Synthase from Antrodia cinnamomea.

Antrodia cinnamomea is an endemic species found in Taiwan, known for its medicinal properties in treating various discomforts, including inflammation, diarrhea, abdominal pain, and other diseases. A. cinnamomea contains terpenoids that exhibit numerous bioactivities, making them potential food additives. This discovery piqued our interest in uncovering their biosynthetic pathway. Herein, we conducted functional and structural characterization of a sesquiterpene synthase Cop4 from A. cinnamomea (AcCop4). Through gas chromatography-mass spectrometry analysis, we observed that AcCop4 catalyzes the cyclization of farnesyl pyrophosphate (FPP), primarily producing cubebol. Cubebol is widely used as a long-lasting cooling and refreshing agent in the food industry. The structure of AcCop4, complexed with pyrophosphate and magnesium ions, revealed the closure of the active site facilitated by R311. Interestingly, binding of pyrophosphate and magnesium ions did not cause any significant conformational change in the G1/2 helix of AcCop4, indicating that the apo form is not fully open. This high-resolution structure serves as a solid basis for understanding the biosynthetic mechanism of AcCop4 and supports further production and modification of cubebol for its applications in the food industry.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Efficient production of Antrodin C by microparticle-enhanced cultivation of medicinal mushroom Antrodia cinnamomea.

The microparticle-enhanced cultivation (MPEC) was used to enhance the production of Antrodin C by submerged fermentation of medicinal mushroom Antrodia cinnamomea. The crucial factors such as types, sizes, concentrations, and addition time of microparticles were optimized. The mechanism of MPEC on the membrane permeability and fluidity of A. cinnamomea and the expression of key genes in Antrodin C were investigated. When talc (18 µm, 2 g/L) was added into the fermentation liquid at 0 h, the promoting effect on Antrodin C was the best. The maximum yield of Antrodin C was 1615.7 mg/L, which was about 2.98 times of the control (541.7 mg/L). Talc slightly damaged the mycelia of A. cinnamomea, increased the release of intracellular constituents, and enhanced the index of unsaturated fatty acid. In addition, the key genes (IDI, E2.3.3.10, HMGCR, atoB) that might play an important role in the synthesis of the triquine-type sesquiterpene Antrodin C, were upregulated. In conclusion, talc increased the permeability and fluidity of cell membrane, upregulated the key genes and improved the biosynthesis process to enhance the yield of Antrodin C in the submerged fermentation of A. cinnamomea.

Antrodia camphorata polysaccharide improves inflammatory response in liver injury via the ROS/TLR4/NF-κB signal.

Antrodia Camphorata Polysaccharide (ACP) refers to a kind of polysaccharide extracted from the natural porous fungus Antrodia camphorata. This study investigated the mechanism of action of ACP in protecting the liver. The results showed that ACP suppressed the LPS-induced KC cell activation, reduced the expression of inflammatory factors, increased the SOD level and suppressed ROS expression. In addition, N-acetylcysteine (NAC) was adopted for pre-treatment to suppress ROS. The results indicated that NAC synergistically exerted its effect with ACP, suggesting that ACP played its role through suppressing ROS. Further detection revealed that ACP activated the Nrf2 signal. It was discovered in the mouse model that, ACP effectively improved liver injury in mice, decreased ALT and AST levels, and suppressed the expression of inflammatory factors. This study suggests that ACP can exert its effect against oxidative stress via the Nrf2-ARE signalling, which further improves the production of ROS and the activation of TLR4-NF-κB signalling, and protects the liver against liver injury.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

Structural sequencing and anti-inflammatory, anti-lung cancer activities of 1,4-α/ß-sulfomalonoglucan in Antrodia cinnamomea.

Antrodia cinnamomea is a precious Polyporaceous fungus with various bioactivities. This study reports the chemical identification and biological activities of sulfomalonoglucan, a sulfated polysaccharide (SPS), from the sodium sulfate enriched medium of the title fungus. The SPS-containing fraction was separated by gel filtration chromatography (GFC) to give the title SPS (denoted as Na10_SPS-F3). By analyzing the evidence for key inter-glycosidic linkages in the 1D and 2D NMR spectroscopic data, one possible repeat unit was proposed as: Na10_SPS-F3 inhibited the secretion of tumor necrosis factor (TNF-α) and interleukin (IL)-6 after lipopolysaccharide (LPS) stimulation in RAW264.7 macrophages. Mechanistically, Na10_SPS-F3 downregulated TGFRII also attenuated the LPS-induced IκB-α degradation. Moreover, Na10_SPS-F3 inhibited lung cancer cell H1975 EGFR/ERK signaling. This is the first paper reporting a 3-O-sulfomalonyl glucan (Na10_SPS-F3) with eight 1,4-ß-Glc moieties connected with ten 1,4-α-Glc moieties from Antrodia cinnamomea and its anti-inflammatory and anti-cancer activities.

Oxidative Stress Induction Is a Rational Strategy to Enhance the Productivity of Antrodia cinnamomea Fermentations for the Antioxidant Secondary Metabolite Antrodin C.

Antioxidant metabolites contribute to alleviating oxidative stress caused by reactive oxygen species (ROS) in microorganisms. We utilized oxidative stressors such as hydrogen peroxide supplementation to increase the yield of the bioactive secondary metabolite antioxidant antrodin C in submerged fermentations of the medicinal mushroom Antrodia cinnamomea. Changes in the superoxide dismutase and catalase activities of the cells indicate that ROS are critical to promote antrodin C biosynthesis, while the ROS production inhibitor diphenyleneiodonium cancels the productivity-enhancing effects of H2O2. Transcriptomic analysis suggests that key enzymes in the mitochondrial electron transport chain are repressed during oxidative stress, leading to ROS accumulation and triggering the biosynthesis of antioxidants such as antrodin C. Accordingly, rotenone, an inhibitor of the electron transport chain complex I, mimics the antrodin C productivity-enhancing effects of H2O2. Delineating the steps connecting oxidative stress with increased antrodin C biosynthesis will facilitate the fine-tuning of strategies for rational fermentation process improvement.

An increase in cell membrane permeability in the in situ extractive fermentation improves the production of antroquinonol from Antrodia camphorata S-29.

The goals of this study were to increase the production of antroquinonol (AQ) and to elucidate the response mechanism of the cell membrane during the in situ extractive fermentation (ISEF) of Antrodia camphorata S-29. Through ISEF, the concentration of AQ reached a maximum of 146.1 ± 2.8 mg/L, which was approximately (7.4 ± 0.1)-fold that of the control (coenzyme Q0-induced fermentation). Transcriptome sequencing showed that four genes (FAD2, fabG, SCD, and FAS1) related to fatty acid biosynthesis were upregulated. FAD2 and SCD may regulate the increase in oleic acid (C18:1) and linoleic acid (C18:2) in the cell membrane of A. camphorata S-29, resulting in an increase in cell membrane permeability. AQ was successfully transferred to the n-tetradecane phase through the cell membrane, reducing product feedback inhibition and improving the production of AQ from A. camphorata S-29.

Enhancement of antroquinonol and antrodin C productions via in situ extractive fermentation of Antrodia camphorata S-29.

This study describes the application of in situ extractive fermentation (ISEF) to increase the yields of antroquinonol (AQ) and antrodin C (AC) from Antrodia camphorata S-29. In initial screening experiments, nine solvents were tested to identify the most suitable extractant for the in situ extraction of AQ and AC. These solvents included n-tetradecane, n-dodecane, n-decane, heavy paraffin, light paraffin, oleyl alcohol, oleic acid, butyl oleate, and isopropyl myristate. Of these, oleic acid was the most suitable solvent for the in situ extraction of AQ and AC. The use of oleic acid as an in situ extractant significantly improved AQ and AC productions, which were approximately 5-fold and 8-fold that of the control, respectively. The recovered oleic acid was treated with a silica gel solid-phase extraction column, which was able to rapidly adsorb the bioactive metabolites. The separated solvent hardly contained fermentation products and could be directly reused in ISEF. AQ and AC were obtained with purities of over 75% by silica gel column chromatography. The recoveries of AQ and AC reached 70.7 ± 0.8% and 81.5 ± 1.2%, respectively.

Diversity of potentially exploitable pharmacological activities of the highly prized edible medicinal fungus Antrodia camphorata.

Antrodia camphorata, also known as A. cinnamomea, is a precious medicinal basidiomycete fungus endemic to Taiwan. This article summarizes the recent advances in research on the multifarious pharmacological effects of A. camphorata. The mushroom exhibits anticancer activity toward a large variety of cancers including breast, cervical, ovarian, prostate, bladder, colorectal, pancreatic, liver, and lung cancers; melanoma; leukemia; lymphoma; neuroblastoma; and glioblastoma. Other activities encompass antiinflammatory, antiatopic dermatitis, anticachexia, immunoregulatory, antiobesity, antidiabetic, antihyperlipidemic, antiatherosclerotic, antihypertensive, antiplatelet, antioxidative, antiphotodamaging, hepatoprotective, renoprotective, neuroprotective, testis protecting, antiasthmatic, osteogenic, osteoprotective, antiviral, antibacterial, and wound healing activities. This review aims to provide a reference for further development and utilization of this highly prized mushroom.

Study on antrodia camphorata polysaccharide in alleviating the neuroethology of PD mice by decreasing the expression of NLRP3 inflammasome.

Parkinson's disease (PD) is a neurodegenerative disease, and the role of neuroinflammation in the pathogenesis and progression of PD has been confirmed. The polysaccharides and triterpenoids of antrodia camphorata (a polyporous fungus) harbor diverse and powerful pharmacological effects. In this study, 6-hydroxydopamine was used to construct a PD mouse model. After antrodia camphorata polysaccharide (ACP) intervention, neurobehavioral changes were detected, neurotransmitter changes in striatum were determined by high-performance liquid chromatography, the alterations of striatal NOD-like receptor pyrin domain containing three (NLRP3) were examined by immunohistochemistry, and the expression of NLRP3, IL-1ß, Caspase-1, and proCaspase-1 were detected by western blot. To be specific, the items of neurobehavioral test included open field activity, rotary test, pole test, gait analysis, and swimming test. As a result, 6-hydroxydopamine could lead to PD-like lesions, including tremor, stiffness, attenuated spontaneous activity, and bradykinesia in mice, and the expression of tyrosine hydroxylase in the striatum was decreased. After ACP intervention, the neuroethology of mice was significantly improved, as demonstrated by the elevated levels of dopamine in the striatum and the decreased expression of dopamine in the striatum in NLRP3 inflammasome. NLRP3 inflammasome played an important role in neuroinflammation in PD mice. ACP could reduce the activation of NLRP3 and expression of related inflammatory factors.

Sodium thiosulfate enhances production of polysaccharides and anticancer activities of sulfated polysaccharides in Antrodia cinnamomea.

Sulfated polysaccharides (SPSs) are polysaccharides (PSs) with high sulfate functionalization and possess bioactivities. This study aimed to increase the sulfate content of SPSs in Antrodia cinnamomea through sulfate feeding. Feeding A. cinnamomea with sodium thiosulfate was found to increase yields of PSs and SPSs in A. cinnamomea. The SPSs thus obtained (ST-SPS) were further isolated, showing enhanced sulfate content of 2.5 mmol/g. Sodium thiosulfate induced changes in molecular weight from 320 kDa to 1342 kDa, and area percentage of low-molecular-weight ST-SPS (< 20 kDa) was decreased. Functional studies revealed that sodium thiosulfate increased the ST-SPS anticancer efficacy in cancer cells via inhibition of EGFR/AKT signaling. Moreover, the ST-SPS enhanced synergistically cisplatin-, gefitinib- and 5 FU-induced cytotoxic effects in lung cancer H1975 cells and colon cancer CT26 cells. This study is the first to demonstrate that sodium thiosulfate induced changes in properties of A. cinnamomea with the anticancer mechanisms of ST-SPS.

4-Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

Enhancement of antroquinonol production during batch fermentation using pH control coupled with an oxygen vector.

BACKGROUND: Antroquinonol, a ubiquinone derivative that shows anticancer and anti-inflammatory activities, is produced during solid-state fermentation of Antrodia camphorata; however, it cannot be biosynthesized via conventional submerged fermentation. RESULTS: A method for enhancing the biosynthesis of antroquinonol by controlling pH and adding an oxygen vector in a 7 L bioreactor was studied. In shake-flask experiments, a maximum antroquinonol production of 31.39 ± 0.78 mg L-1 was obtained by fermentation with adding 0.2 g L-1 coenzyme Q0 (CoQ0 ), at the 96th hour. Following kinetic analysis of the fermentation process, pH control strategies were investigated. A maximum antroquinonol production of 86.47 ± 3.65 mg L-1 was achieved when the pH was maintained at 5.0, which exhibited an increase of 348.03% higher than the batch without pH regulation (19.30 ± 0.88 mg L-1 ). The conversion rate of CoQ0 improved from 1.51% to 20.20%. Further research revealed that the addition of n-tetradecane could increase the production of antroquinonol to 115.62 ± 4.87 mg L-1 by increasing the dissolved oxygen in the fermentation broth. CONCLUSION: The results demonstrated that pH played an important role in antroquinonol synthesis in the presence of the effective precursor CoQ0 . It was a very effective strategy to increase the yield of antroquinonol by controlling pH and adding oxygen vector. © 2018 Society of Chemical Industry.

Production, structure and morphology of exopolysaccharides yielded by submerged fermentation of Antrodia cinnamomea.

Carbon and nitrogen sources in culture medium of Antrodia cinnamomea were optimized to eliminate the interference of exterior macromolecules on exopolysaccharide (EPS) yield by submerged fermentation. The results suggested that culture medium containing 50 g/L of glucose and 20 g/L of yeast extract as the optimal carbon and nitrogen sources could produce 1.03 g/L of exopolysaccharides. After purification, two heteropolysaccharides (AC-EPS1 and AC-EPS2) were obtained and characterized to provide the basic structure information. As the main component of the produced EPS, AC-EPS2 (accounting for 89.63%) was mainly composed of galactose (87.42%) with Mw (molecular weight) and R.M.S. (root-mean-square) radius of 1.18 × 105 g/mol and 25.3 nm, respectively. Furthermore, the spherical and flexible chain morphologies of EPS were observed in different solvents by TEM. The structural and morphological information of purified EPS were significant for further study on their structure-activity relationship and related applications.

Alpha-terpineol affects synthesis and antitumor activity of triterpenoids from Antrodia cinnamomea mycelia in solid-state culture.

To enhance production of Antrodia cinnamomea triterpenoids (ACTs) from mycelia in solid-state culture, α-terpineol was added to the medium as an elicitor at an optimal concentration of 0.05 mL L-1. Multi-stage solvent extraction and HPLC analysis were performed, and the compositions of ACTs-E (from culture with elicitor) and ACTs-NE (from culture without elicitor) were found to be quite different. In assays of in vitro antitumor activity, ACTs-E, in comparison with ACTs-NE, produced stronger viability reduction in several tumor cell lines and stronger apoptosis induction in HeLa in a dose-dependent manner. Several related proteins involved in the mitochondrial pathway of apoptosis (p53, Bax, caspase-3) did not show expression upregulation by ACTs-E, suggesting that apoptosis induction occurred through a p53-independent process. Further analysis revealed that ACTs-E strongly inhibited synthesis of topoisomerase I (TOP1) and tyrosyl-DNA phosphodiesterase I (TDP1), which are involved in DNA repair, at both transcriptional and protein levels. Our findings suggest that ACTs-E have potential for applications in the pharmaceutical, clinical, and functional food industries, as a novel antitumor agent and a dual TOP1/TDP1 inhibitor.

Amelioration effects of nanoencapsulated triterpenoids from petri dish-cultured Antrodia cinnamomea on reproductive function of diabetic male rats.

PURPOSE: Nanoencapsulated triterpenoids from petri dish-cultured Antrodia cinnamomea (PAC) and its amelioration effects on reproductive function in diabetic rats were investigated. MATERIALS AND METHODS: PAC encapsulated in silica-chitosan nanoparticles (Nano-PAC) was prepared by the biosilicification method. The diabetic condition in male Sprague Dawley rats was induced by high-fat diet and streptozotocin (STZ). Three different doses of Nano-PAC (4, 8, and 20 mg/kg) were administered for 6 weeks. Metformin and control of nanoparticles (Nano-con) were taken as positive and negative controls, respectively. RESULTS: The average particle size was ~79.46±1.63 nm, and encapsulation efficiency was ~73.35%±0.09%. Nano-PAC administration improved hyperglycemia and insulin resistance. In addition, Nano-PAC ameliorated the morphology of testicular seminiferous tubules, sperm morphology, motility, ROS production, and mitochondrial membrane potential. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) antioxidant, as well as testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were increased, whereas proinflammatory cytokines TNF-α, IL-6, and IFN-γ were decreased. CONCLUSION: In the present study, we successfully nanoencapsulated PAC and found that a very low dosage of Nano-PAC exhibited amelioration effects on the reproductive function of diabetic rats.

Antrodia cinnamomea mycelial fermentation broth inhibits the epithelial-mesenchymal transition of human esophageal adenocarcinoma cancer cells.

Esophageal cancer is associated with a high mortality rate and easy metastasis. The aim of this study is to investigate the effect of the bio-product Antrodia cinnamomea mycelial fermentation broth (AC-MFB) on the epithelial mesenchymal transition (EMT) of human esophageal cancer cells and the molecular mechanisms underlying these effects. Transforming growth factor ß (TGF-ß) was used to induce EMT in human esophageal BE3 cancer cells. Changes in cell morphology and migration potential were examined. The expression of E-cadherin, N-cadherin, vimentin, and other transcriptional factors was studied by western blot assay. The results showed that AC-MFB was not only able to upregulate the expression of Ecadherin and attenuate the TGF-ß-induced overexpression of vimentin and N-cadherin, but it also reversed the TGF-ß-induced changes in cell morphology from polygonal to spindle-shaped and delayed the migration potential of BE3 cells. Furthermore, AC-MFB treatment was able to inhibit the expression levels of both Twist and Twist1. Overall, AC-MFB was able to inhibit the EMT of esophageal cancer BE3 cells, which was accompanied by Twist and Twist1 downregulation.

Current Advances on the Structure, Bioactivity, Synthesis, and Metabolic Regulation of Novel Ubiquinone Derivatives in the Edible and Medicinal Mushroom Antrodia cinnamomea.

In recent years, Antrodia cinnamomea has attracted great attention around the world as an extremely precious edible and medicinal mushroom. Ubiquinone derivatives, which are characteristic metabolites of A. cinnamomea, have shown great bioactivities. Some of them have been regarded as promising therapeutic agents and approved into clinical trial by the U.S. Food and Drug Administration. Although some excellent reviews have been published covering different aspects of A. cinnamomea, this review brings, for the first time, complete information about the structure, bioactivity, chemical synthesis, biosynthesis, and metabolic regulation of ubiquinone derivatives in A. cinnamomea. It not only advances our knowledge on the bioactive metabolites, especially the ubiquinone derivatives, in A. cinnamomea but also provides valuable information for the investigation on other edible and medicinal mushrooms.