Oral exosome-like nanovesicles from Phellinus linteus suppress metastatic hepatocellular carcinoma by reactive oxygen species generation and microbiota rebalancing.

The biomedical application of nanotechnology in cancer treatment has demonstrated significant potential for improving treatment efficiencies and ameliorating adverse effects. However, the medical translation of nanotechnology-based nanomedicines faces challenges including hazardous environmental effects, difficulties in large-scale production, and possible excessive costs. In the present study, we extracted and purified natural exosome-like nanoparticles (ELNs) from Phellinus linteus. These nanoparticles (denoted as P-ELNs) had an average particle size of 154.1 nm, displayed a negative zeta potential of -31.3 mV, and maintained stability in the gastrointestinal tract. Furthermore, P-ELNs were found to contain a diverse array of functional components, including lipids and pharmacologically active small-molecule constituents. In vitro investigations suggested that they exhibited high internalization efficiency in liver tumor cells (Hepa 1-6) and exerted significant anti-proliferative, anti-migratory, and anti-invasive effects against Hepa 1-6 cells. Strikingly, the therapeutic outcomes of oral P-ELNs were confirmed in an animal model of metastatic hepatocellular carcinoma by amplifying reactive oxygen species (ROS) and rebalancing the gut microbiome. These findings demonstrate the potential of P-ELNs as a promising oral therapeutic platform for liver cancer treatment.

Inhibitory Effect of Styrylpyrone Extract of Phellinus linteus on Hepatic Steatosis in HepG2 Cells.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 µg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1ß) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Phelliribsin B: A New Styrylpyrone Derivative from the Medicinal Fungus Phellinus ribis.

A novel styrylpyrone derivative, named phelliribsin B (1), as well as four biogenetically related known compounds, phellifuropyranone A (2), inoscavin C (3), inoscavin A (4), and inoscavin D (5) were separated and purified from the medicinal fungus Phellinus ribis. The structure of phelliribsin B was determined by spectroscopic analysis, and the absolute configuration was assigned by experimental and calculated ECD data. Additionally, the plausible biosynthetic pathway of 1 was also proposed. Compound 1 showed moderately cytotoxic activity against HepG2 and SKOV-3 tumor cell lines with IC50 values of 32.71 and 57.89 µM, respectively. Based on the results of cytotoxicity against HepG2 tumor cells, the structure-activity relationship of compounds 1-4 with similar skeletons was discussed. The styrylpyrone derivatives with similar skeletons have moderately cytotoxic activity and have the potential to play an important role in the anti-tumor treatment.

Selected Species of the Genus Phellinus - Chemical Composition, Biological Activity, and Medicinal Applications.

This study presents the current knowledge on chemical composition, biological activity, and possible medicinal applications of Phellinus igniarius, Phellinus pini, Phellinus pomaceus, and Phellinus robustus. These inedible arboreal species are phytopathogens that cause the enzymatic decomposition of wood. These species belong to the medicinal mushrooms and have been known for centuries in the traditional medicine of the Far East. They have been used as an effective remedy for stomach and intestinal ailments, diarrhea, and hemorrhages. Mycochemical studies have proved the presence of polysaccharides, phenolic compounds, and terpenoids. These compounds show biological activities such as anticancer, antioxidant, antiangiogenic, and antiviral. Research studies conducted using modern analytical methods have advanced the knowledge on the potential therapeutic use of compounds isolated not only from the fruiting bodies but also from biomass obtained with in vitro biotechnological methods.

Aromatic Cadinane Sesquiterpenoids from the Fruiting Bodies of Phellinus pini Block SARS-CoV-2 Spike-ACE2 Interaction.

The ongoing COVID-19 global pandemic caused by SARS-CoV-2 inspires the development of effective inhibitors to block the SARS-CoV-2 spike-ACE2 interaction. A chemical investigation on the fruiting bodies of Phellinus pini led to the isolation of five aromatic cadinane sesquiterpenoids including four new ones, named piniterpenoids A-D (1-4), as well as three known lignans. Their structures were determined by extensive spectroscopic analysis including HRMS and 1D and 2D NMR. All of the aromatic cadinane sesquiterpenoids inhibited the SARS-CoV-2 spike-ACE2 interaction, with IC50 values ranging from 64.5 to 99.1 µM. A molecular docking study showed the disruption of the interaction of compound 1 via hydrogen interactions with Arg403, Asp405, and Arg408 of SARS-CoV-2 RBD and Arg393 and His34 residues of ACE2. These results suggested that aromatic cadinane sesquiterpenoids might be useful in developing agents for COVID-19.

Biological activities of two polypore macrofungi (Basidiomycota) and characterization of their compounds using HPLC-DAD and LC-ESI-MS/MS.

Members of Hymenochaetaceae fungi are among well-known macromycetes with various medicinal properties. The aim of this study was to investigate the biological activities of Phellinus tuberculosus and Fuscoporia ferruginosa collected in Iran. The antimicrobial, antioxidant, and cytotoxic activities of the two species were examined, and their phenolic and polysaccharide contents were quantified. Compounds were characterized by HPLC-DAD chromatography and LC-ESI-MS/MS spectroscopy. According to our results, the antibacterial and antioxidant effects of P. tuberculosus extracts were stronger than F. ferruginosa. Also, the effect of hydroalcoholic extracts was higher than the aqueous extract. Gram-positive bacteria were more sensitive to all extracts, especially Streptococcus mutans with a MIC of 0.7 mg/mL and MBC of 6.25 mg/mL. HPLC-DAD analyses detected gallic acid, caffeic acid, and syringic acid in both fungi. The LC-ESI-MS/MS confirmed the detected compounds in HPLC-DAD and showed the presence of several phenolic compounds such as phellifuropyranone, phelligridin, and hispidin, besides others. This study showed that F. ferruginosa and P. tuberculosus are potent medicinal fungi with antibacterial and antioxidant properties, with no toxic effect on normal HDF cells, and possess various bioactive compounds including styrylpyrone-type phenols with well-known bioactivities.

Screening anti-gout compounds from Phellinus igniarius by ultrafiltration liquid chromatography mass spectrometry based on evaluation of an in vitro method combined with enzymatic reaction.

In the present study, the anti-inflammation effect of Phellinus igniarius extract was detected on an in vitro model of RAW 264.7 cells stimulated using sodium urate. In this cell model, the content changes of inflammatory cytokines, intercellular adhesion molecule-1, and interleukin-1 beta, in cell culture supernatants were detected using an enzyme-linked immunosorbent assay. The xanthine oxidase inhibitory activity of P. igniarius extracts was determined using a microplate reader. Furthermore, in order to identify the active compounds of P. igniarius, ultrafiltration liquid chromatography mass spectrometry was utilized to screen xanthine oxidase inhibitors from the extract. Our results showed that in the presence of P. igniarius extract, the expressions of interleukin-1 beta and intercellular adhesion molecule-1 decreased (p < 0.01 and p < 0.05, respectively) compared to that in the control group. The extract effective inhibited the xanthine oxidase activity. Finally, seven compounds were screened and identified as potential xanthine oxidase inhibitors from P. igniarius. Taken together, these results demonstrate a potential anti-inflammation bioactivity of P. igniarius in vitro, providing a basis for further in vivo research for the prevention and treatment of gout.

Preparation and characterization of selenium-rich polysaccharide from Phellinus igniarius and its effects on wound healing.

The modified of polysaccharides show various bio-activities. In our work, Phellinus igniarius Selenium-enriched mycelias polysaccharides (PSeP) were prepared from Phellinus igniarius, and its antioxidant and anti-inflammatory effects on injured mice were evaluated. The selenium content and physical properties of polysaccharides were characterized by GC, HPGPC, and FT-IR analysis. The results showed that PSeP could reduce reactive oxygen species (ROS) levels, myeloperoxidase (MPO) activity as well as malondialdehyde (MDA) content. Meanwhile, it increased the enzyme activities of glutathione peroxidase (GSH-Px) and catalase (CAT). Finally, it showed obvious wound healing effects in vivo. Moreover, PSeP could clear the ROS without obvious cytotoxicity. PSeP could further improve its ability to clear ROS level to promote skin wound healing in mice three days in advance.

Anti-Inflammatory Properties In Vitro and Hypoglycaemic Effects of Phenolics from Cultivated Fruit Body of Phellinus baumii in Type 2 Diabetic Mice.

Dietary intervention in type 2 diabetes mellitus (T2DM) is a hotspot in international research because of potential threats to human health. Phellinus baumii, a wild fungus traditionally used as a food and medicine source, is now cultivated in certain East Asian countries, and is rich in polyphenols, which are effective anti-inflammatory ingredients useful in treatment of T2DM, with fewer side effects than drugs. To examine the hypoglycaemic effects of Phellinus baumii phenolics (PPE), the metabolite profiles of T2DM mice induced by streptozotocin after PPE intervention were systematically analyzed. Here, 10 normal mice were given normal saline as control group, and 50 model mice were randomly assigned to five groups and daily intragastric administrated with saline, metformin (100 mg/kg), and PPE (50, 100, 150 mg/kg of body weight), for 60 days. The pro-inflammatory factor contents of lipopolysaccharide stimulation of RAW 264.7 cells were decreased in a dose-dependent manner after PPE treatment, we propose that PPE could exert anti-inflammatory properties. PPE could also effectively reduce blood glucose levels, increased insulin sensitivity, and improved other glucolipid metabolism. Q-PCR results suggested that the hypoglycemic effects of PPE might be through activating IRS1/PI3K/AKT pathway in diabetic mice. These results suggest that PPE has strong potential as dietary components in the prevention or management of T2DM.

Separation, characterization and hypoglycemic activity in vitro evaluation of a low molecular weight heteropolysaccharide from the fruiting body of Phellinus pini.

Edible mushrooms have potential in anti-diabetic phytotherapy. They are rich in natural compounds such as polysaccharides, which have been known to have antihyperlipidemic effects since ancient times. A polysaccharide fraction of PP80 and a contained low molecular-weight (Mw), water-soluble polysaccharide (PPW-1, Mw: 3.2 kDa) were isolated from the fruiting body of Phellinus pini. Both PP80 and PPW-1 possess α-glucosidase inhibition and glucose consumption amelioration in an insulin-resistant HepG2 cell model. The α-glucosidase inhibitory activity of PPW-1 (IC50 = 2.2 ± 0.1 mg mL-1) is significantly (P < 0.01) higher than those of PP80 (IC50 = 13.1 ± 0.5 mg mL-1) and acarbose (IC50 = 4.3 ± 0.2 mg mL-1), behaving in a non-competitive inhibition manner. The structural characterization results indicated that PPW-1 is a homogeneous heteropolysaccharide composed of d-glucose, d-mannose, d-galactose and l-rhamnose. The major backbone of PPW-1 is primarily comprised of 1,6-linked glucopyranose, every third residue of which is branched at the O-3 position by a side chain consisting of 1,3-linked and terminal glucopyranose. In addition, small amounts of 1,2-linked-α-d-Manp, 1,6-linked-3-O-Me-α-d-Galp and rhamnose exist in PPW-1. In summary, PPW-1 is a novel heteropolysaccharide with potent in vitro hypoglycemic activity, and it may be a potential dietary component for improving glucose homeostasis.

Enhanced Mycelium Production of Phellinus igniarius (Agaricomycetes) Using a He-Ne Laser with Pulsed Light.

This study used a He-Ne laser with pulsed light irradiation to produce mutant strains of Phellinus igniarius strain JQ9 with enhanced characteristics for fermentation (17.685 ± 3.092 g/L) compared with the parent strain (12.062 ± 1.119 g/L). The combined treatment conditions were as follows: He-Ne laser irradiation for 30 min using a spot diameter of 10 mm, pulsed light treatment power set at 100 J, a treatment distance of 14.5 cm, and a flash frequency of 0.5 s. The production of bioactive polysaccharides and small biocompounds such as polyphenols, flavonoids, and triterpenes increased together with mycelium production. The results showed that polyphenol content was significantly correlated with L*, a*, and b* values (R = -0.594, P < 0.01; R = 0.571, P < 0.01; and R = 0.500, P < 0.05; respectively). Antagonistic and random amplified polymorphic DNA analyses indicated that the genetic material of the screened mutants was altered. The mutant screening using a He-Ne laser with pulsed light irradiation could be an effective method for the development of Phellinus strains and could thus improve mycelium production.

Development of ultrasound-assisted mixture extraction and online extraction solution concentration coupled with countercurrent chromatography for the preparation of pure phytochemicals from Phellinus vaninii.

Herein, ultrasound-assisted mixture extraction (UAME) and online extraction solution concentration (OESC) were conducted to extract products from crops and plants. These techniques were coupled with parallel countercurrent chromatography (PCCC) and applied for continuous extraction and online isolation of chemical constituents from Phellinus vaninii. The UAME instrument comprises extraction and solution separation chambers. It provides higher extraction efficiency and fewer impurities and is suitable for processing various sample matrices. The OESC device comprises a spray nozzle, concentrating cylinder, and hot-blast air nozzle. The mechanical parameters for UAME and OESC were optimized, and the operation of online UAME and OESC coupled with PCCC was described. Raw plant materials were extracted using a two-phase extractant comprising petroleum-ethyl acetate-ethanol-water (0.5:2.0:0.5:2.0, v/v/v/v). The aqueous and organic phases were then concentrated using the OESC technique. Two CCC runs were conducted for preparatory work. After extraction and online concentration, the concentrate was pumped into the CCC for separation. During PCCC separation, continuous automated extraction and concentration were still conducted. When the first cycle of the UAME/OESC/PCCC was completed, followed by the initiation of the second cycle, and the process was continued. Six target compounds with purities exceeding 97.22% were successfully separated using the CCC solvent systems comprising n-hexane-ethyl acetate-acetonitrile-water (5.5:2.5:5.0:0.4, v/v/v/v) and n-butanol-ethanol-water (4.5:1.3:6.5, v/v/v). Compared with conventional extraction methods, the proposed UAME/OESC/PCCC method has higher efficiency, facilitates high-purity separation of analytes, and offers opportunity for automation and systematic preparation of natural products.

The phytochemistry and pharmacology of medicinal fungi of the genus Phellinus: a review.

Phellinus Quél is one of the largest genera of Hymenochaetaceae, which is comprised of about 220 species. Most Phellinus macro-fungi are perennial lignicolous mushrooms, which are widely distributed on Earth. Some Phellinus fungi are historically recorded as traditional medicines used to treat various diseases in eastern Asian countries, especially China, Japan and Korean. Previous phytochemical studies have revealed that Phellinus fungi produce diverse secondary metabolites, which mainly contain polysaccharides, flavones, coumarins, terpenes, steroids, and styrylpyranones. Pharmacological documents have demonstrated that Phellinus mushrooms and their compounds have a variety of bioactivities, such as anti-tumor, immunomodulation, anti-oxidative and anti-inflammation, anti-diabetes, neuro-protection, and anti-viral effects. This review surveys the literature reporting the isolation, characterization, and bioactivities of secondary metabolites from the fungi of the genus Phellinus, focusing on studies published in the literature up to April 2020. Herein, a total of more than 300 compounds from 13 Phellinus species and their isolation, characterization, chemistry, pharmacological activities, and relevant molecular mechanisms are comprehensively summarized.

Active ingredients and mechanisms of Phellinus linteus (grown on Rosa multiflora) for alleviation of Type 2 diabetes mellitus through network pharmacology.

Phellinus linteus (mushroom) grown on Rosa multiflora (PL@RM), exposed beneficial effect and safety on Type 2 diabetes mellitus (T2DM) from Korean folk remedies. However, its active chemical constituents and mechanism(s) against T2DM have not been confirmed. Hence, we deciphered the active compounds and mechanism(s) of PL@RM against T2DM through network pharmacology. GC-MS of PL@RM manifested 54 compounds and drug-likeness properties of these compounds were confirmed by Lipinski's rule. The compound (40) related genes were composed of Similarity Ensemble Approach (SEA) and SwissTargetPrediction (STP). The overlapping genes (61) between the two databases were identified. Besides, the T2DM related genes (4,736) were extracted from DisGeNet and OMIM database. In parallel, a Venn diagram was constructed between the overlapping genes (61) and T2DM related genes (4,736), and finally, 48 genes were picked. The interactive networks between compounds and overlapping genes were plotted and visualized by RStudio. In addition, KEGG Pathway enrichment analysis was evaluated by String. String analysis showed that the mechanisms of PL@RM against T2DM were related to 16 pathways, where inhibition of gluconeogenesis by inactivating metabolic pathways was noted as the hub pathway of PL@RM against T2DM. Besides, bubble chart indicated that activation of the AMPK signaling pathway might enhance the insulin receptor (IR) phosphorylation, which is regarded the key signaling pathway of PL@RM against T2DM. Furthermore, the autodock vina revealed the promising binding affinity energy of the epicholesterol (the most drug-likeness compound) on HMGCR (hub gene). Overall, this work hints at the therapeutic evidence of PL@RM on T2DM, and this data expound the main chemical compounds and mechanisms of PL@RM against T2DM.

Anti-angiogenic effect of a chemically sulfated polysaccharide from Phellinus ribis by inhibiting VEGF/VEGFR pathway.

Previous studies have demonstrated that the sulfated polysaccharide named PRP-S16 could inhibit the proliferation, migration, and tube formation of endothelial cells in vitro. Here, its anti-angiogenic effect and mechanism in vivo were investigated by Lewis lung carcinoma (LLC) mice model. PRP-S16 significantly reduced the microvessel density (MVD) of tumor, exhibiting a high tumor growth inhibitory effect in LLC mice. All designed assays including quantitative real-time PCR, immunohistochemistry, enzyme-linked immunosorbent assay and western blotting showed that PRP-S16 reduced the mRNA and the protein expression of vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) in serum or tumor tissue of mice. Western blotting also detected decreased phosphorylated (p)-VEGFR-1, p-VEGFR-2, hypoxia-inducible factor-1α (HIF-1α), protein kinase B (Akt), and matrix metalloproteinases-9 (MMP-9). PRP-S16 had no adverse effects on angiogenesis in non-target organs. These findings suggested that the mechanism of anti-angiogenesis of PRP-S16 in vivo was due to inhibition of VEGF/VEGFR signaling pathway and it might be a promising candidate for tumor by anti-angiogenic therapy.

Phellinus baumii enhances the immune response in cyclophosphamide-induced immunosuppressed mice.

Phellinus species is a mushroom used as traditional medicine in Eastern Asia. Research on Phellinus baumii (PB) is relatively limited; however, it has been reported to have antioxidant, DNA damage-protecting, immunostimulating, and antidiabetic activities. In our previous study on anti-inflammatory properties in lipopolysaccharide-stimulated RAW 264.7 cells and the various bioactive components of PB, we propose that PB could exert immune enhancing effects. Therefore, our current study aimed to investigate the immune-enhancing effect on immunosuppressed mice. Different concentrations of PB extract (0, 50, 100, 200, and 400 mg/kg body weight) were given to mice via oral gavage for 6 weeks accompanied by intraperitoneal cyclophosphamide administration to induce immunosuppression. A bone marrow micronucleus test was performed in mice to screen for potential genotoxic compounds. Splenocyte viability and proliferation, splenic and peritoneal natural killer cell activities, and hematological markers were then measured. Cytokines in the spleen and serum, as well as splenic mRNA levels of nuclear factor-κB; interferon-γ; tumor necrosis factor-α; and interleukin (IL)-1ß, IL-6, and IL-12, were determined in mice. As a result, PB ameliorated T- and B-lymphocyte proliferation, splenic and peritoneal NK cell activities, bone marrow cells, hematological markers, cytokine levels, and T-lymphocyte numbers. Moreover, serum and spleen cytokine levels and mRNA expression were elevated in the PB groups compared to controls. Our results suggest that the PB extract can be used as a potent immunomodulator under immunosuppressive conditions. Thus, PB may be used as a potent biofunctional and pharmaceutical material to potentially enhance human immunity.

Hepatoprotective effect of Phellinus linteus mycelia polysaccharide (PL-N1) against acetaminophen-induced liver injury in mouse.

Edible and medicinal fungi are one of the major sources for extraction and identification of polysaccharides, which are important biological response modifiers with notable antitumor, hepatoprotective effect and other pharmacological activities. This study aimed to evaluate the hepatoprotective effect of isolated Phellinus linteus polysaccharide (PL-N1) against acetaminophen (APAP) induced liver injury in mice. Mice were treated intragastrical with PL-N1 (10, 50 and 100 mg/kg) and APAP (300 mg/kg) injection. APAP alone caused increased serum aminotransferase levels and changes in hepatic histopathology, promoted oxidative stress by increasing lipid peroxidation and decreasing anti-oxidant enzyme activities, leading to hepatocellular necrosis and reduced liver function. PL-N1 decreased cytochrome P450 2E1 (CYP2E1) expression and hepatic release of cytokines to enhance the level of phase II enzymes. Also, PL-N1 obviously accelerates the metabolism of APAP in the rat model. Molecular docking analysis revealed the α-d-glucopyranosyl exhibit maximum interaction (-8.099) against CYP2E1 as comparably less than standard drug silibinin (-13.767). PL-N1 could be a promising natural substance for ameliorating acute APAP-induced oxidative stress and hepatic injury.