Pulsed Light Mutagenesis of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), for Enhanced Production of Flavonoids, Laccase, and Fermentation Biomass.

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

Anti-cancer Potential of Polysaccharide Extracted From Polygonatum sibiricum on HepG2 Cells via Cell Cycle Arrest and Apoptosis.

Polygonatum sibiricum is one of the most widely used traditional Chinese medicine in China. Polygonatum sibiricum polysaccharide (PSP) is the main functional component of Polygonatum sibiricum. In this study, a water-soluble polysaccharide (PSP-1) was first isolated from Polygonatum sibiricum with a molecular weight of 38.65 kDa. Structural analysis was performed via methylation and FT-IR spectroscopy analyses, which in combination with NMR spectroscopy, revealed that PSP-1 has a → 4-α-D-Glcp-1 → backbone with the substitution at O-6 with the ß-D-Glcp-1 → residues. Furthermore, PSP-1 exhibited potent and concentration-dependent anticancer effects, inducing HepG2 cell apoptosis and arresting the cell cycle at the G1 phase. Moreover, PSP-1 also decreased the mitochondrial membrane potential, damaged the nucleus of HepG2 cells, and increased the activity of caspase-9 and-3 in the intrinsic apoptotic pathways to induce HepG2 cell apoptosis. To conclude, PSP-1 might be a good candidate for the treatment of liver cancer, and this work provides important information for understanding the relationship between structure and antitumor activity of PSP-1, which is relevant for the treatment of hepatocellular carcinoma in clinic.

Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production.

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

Bronchospasm and Kidney Injury Associated With Salvaged Blood Reinfusion During Pediatric Neurosurgical Procedure: A Case Report.

Adverse events associated with salvaged blood reinfusion are common, but bronchospasm has been rarely reported, especially in pediatrics. We report the case of a child undergoing epileptogenic focus resection, who experienced bronchospasm and kidney injury associated with reinfusion of salvaged blood, presumably related to excessive free hemoglobin.

Structural, rheological properties and antioxidant activities of polysaccharides from mulberry fruits (Murus alba L.) based on different extraction techniques with superfine grinding pretreatment.

The structural characteristics and biological activity of polysaccharides were influenced by different extraction methods. In this study, polysaccharides from mulberry fruits (Murus alba L., which were pre-treated with superfine grinding process) (MFP) were exacted using hot-water extraction (HWE), enzyme-assisted hot water extraction (EAHE), ultrasonic-assisted hot water extraction (UAHE), and high-speed shear homogenization-assisted hot water extraction (HSEHE). The extraction yield, structure, rheological properties and antioxidant activities of MFPs were investigated. MFP extracted using the HSEHE method have the highest extraction yields than other extraction methods. The smaller particle size of mulberry powder was found to improve the extraction yields. The MFPs were obtained by the combination between different extraction methods and superfine grinding pretreatment (through 100 mesh sieve) (MFP-HWE100, MFP-EAHE100, MFP-UAHE100, MFP-HSEHE100) showed the same levels of monosaccharide compositions and glycosyl linkages, However, these methods can produce MFP with different monosaccharide proportions, branching degree, different molecular weight, particle size and microstructure. MFP-HSEHE100 achieved the lowest molecular weight and particle size, which exhibited better thixotropy and antioxidant activities than other MFPs. This study identified that HSEHE was the most suitable extraction method for MFP.

Ultrasonic treatment at different pH values affects the macromolecular, structural, and rheological characteristics of citrus pectin.

Ultrasonic degradation has become a promising strategy for producing modified pectin (MP). In this study, the impact of ultrasonic treatment at various pH values (4.0, 7.0, and 10.0) on the macromolecular, structural and rheological characteristics of citrus pectin was investigated. Results demonstrated that ultrasonic irradiation at the higher pH led to larger reductions in the intrinsic viscosity and weight-average molecular weight of pectin. The degradation kinetics of pectin at different pH values under ultrasound well fitted to a second-order reaction kinetics model. Acoustic cavitation, ß-elimination, and demethylation led to the breakage of glycosidic linkages of side chains and methoxyl groups of pectin, but did not have noticeable influences on the main chain of pectin. The ultrasonic treatment at a high pH led to an apparent change in the rheological characteristics of pectin. Therefore, ultrasonic treatment at various pH values can be developed as a viable means to prepare desirable MP.

Antioxidant and Neuroprotector Influence of Endo-Polyphenol Extract from Magnesium Acetate Multi-Stage Addition in the Oak Bracket Medicinal Mushroom, Phellinus baumii (Agaricomycetes).

The objective of this study was to explore the effect of magnesium acetate (MA) addition on the endo-polyphenol yield by Phellinus baumii and establish a feasible additive strategy. The optimal three-point MA addition strategy (0.05 g/L concentration of MA added at 0 h and 6 h, 0.9 g/L concentration of MA added at 12 h) was employed to obtain maximum endo-polyphenol yield. The maximum endo-polyphenol production was reached at 1.22 g/L, which was 1.39-fold higher than that of the control. Additionally, the endo-polyphenol showed stronger antioxidant activity in vitro compared with the control, including DPPH· scavenging capacity (78.76%) and Trolox equivalent antioxidant capacity (TEAC) (32.28 µmol Trolox/g sample). HPLC analysis showed that the endo-polyphenol production of the crude ethanol extracts was significantly higher than that of the control. Hispidin was isolated and identified from the ethanol extract of the culture mycelia from Ph. baumii with the three-point MA addition strategy. Hispidin showed a strong ability to scavenge DPPH free radicals and TEAC, equivalent to positive (vitamin C) value of 89.41% and 75.98%, respectively. Furthermore, hispidin protected H2O2-induced PC12 cells injured by decreased oxidative stress level. These results indicated that the MA multi-stage addition strategy was dependable, and could be used to develop new natural antioxidants for foods or medicines.

A Review on Linking the Medicinal Functions of Mushroom Prebiotics with Gut Microbiota.

In recent years, gut microbiota have been linked to prevention and treatment of human diseases. Mushrooms are a source of potentially useful prebiotics because they contain polysaccharides, terpenoids, and other bioactive compounds. In the present review, we have summarized the prebiotic effects of mushrooms on gut microbiota in the context of immunological, metabolic, neurological, and cancer-related diseases in the last five years. We propose that mushrooms can not only change the composition of gut microbiota, but also promote secretion of beneficial metabolites. In addition, we point to the effects of host mRNA expression in gut microbiota as a direction of further study. Overall, these provide a background for further studies on the mechanisms of regulation of gut microbiota by mushrooms.

Photothermally triggered cytosolic drug delivery of glucose functionalized polydopamine nanoparticles in response to tumor microenvironment for the GLUT1-targeting chemo-phototherapy.

The success in the application of nanomedicines for tumor therapy is largely dependent on the development of efficient tumor targeting, specific and effective drug delivery systems. Here, through a simple synthetic process, we developed a type of novel glucose transporter 1 (GLUT1)-targeting, tumor microenvironment responsive and near infrared irradiation (NIR) induced cytosolic drug delivery nanoparticles (NPs). Our design was based on polydopamine (PDA) NPs as the photothermal agent and drug delivery carrier, glucosyl functional ligands as the GLUT1 targeting agents, and the conjugation of anticancer drug bortezomib (BTZ) to the catechol groups of PDA NPs in a pH-dependent manner. The in vitro and in vivo studies demonstrated that the functionalized PDA NPs could efficiently accumulate in tumor site and localize in subcellular endo/lysosomes of tumor cells, and they could respond to tumor microenvironment and endo/lysosomal pH as well as NIR to promote the robust release of BTZ. Furthermore, the functionalized PDA NPs were first demonstrated to overcome the endo/lysosomal barrier for the enhanced cytosolic BTZ drug delivery through NIR-triggered endo/lysosomal release, achieving the integration of NIR-triggered photothermal effect and chemotherapy for synergistic tumor ablation. The significant suppression and even complete regression of 4 T1 tumor was observed in mice given only single treatment. Therefore, the GLUT1-targeting, pH and photothermal responsive drug delivery NPs show a great potential for broadly applicable chemo-photothermal tumor therapy.

Increased Inhibition Effect of Antrodin C from the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes), on A549 through Crosstalk between Apoptosis and Autophagy.

Antrodin C was obtained from Taiwanofungus camphoratus mycelia. The inhibition effect of antrodin C on A549 lung adenocarcinoma cells was evaluated by plate clone formation, wound healing, cell cycle, activated caspase-3, Bax, P53, Bcl-2, and RAPR activities as well as reactive oxygen species release. Plate clone formation assay revealed that antrodin C could significantly inhibit the viability of A549 cells in vitro. Wound healing assay revealed that cell migration was inhibited by exposure to antrodin C at concentrations of 50 and 80 µg/mL. Flow cytometry revealed that antrodin C increased the percentages of cells in the G0/G1 phase at concentrations of 50 and 80 µg/mL and the apoptosis was related to upregulation of caspase-3, Bax, P53 expression, downregulation of Bcl-2, RAPR expression, and the release of reactive oxygen species in the A549 cells. CQ or RAPA could significantly promote or inhibit the inhibition effect on A549 proliferation induced by antrodin C, which suggests that the autophagy played a cytoprotective role on inhibition proliferation of A549 induced by antrodin C. These results indicated that the combination of pro-apoptosis agents and anti-autophagy agents may be a useful strategy in enhancing the anticancer efficacy in non-small cell lung cancer.

The integration of metabolome and proteome reveals bioactive polyphenols and hispidin in ARTP mutagenized Phellinus baumii.

Phellinus baumii, also called "Sang Huang" in China, is broadly used as a kind of health food or folk medicine in Asia for its high biological activities, e.g. anti-tumor, anti-oxidation and anti-inflammatory activities. Although some previous studies have indicated that polysaccharides and flavonoids showed the activity of inhibiting tumor cells, the active metabolites of P. baumii needs further research. In our study, a stable P. baumii mutant (A67), generated by ARTP mutagenesis strategy, showed more significantly inhibiting tumor cells and enhancing antioxidant activity. Our further studies found that the increase of polyphenols content, especially hispidin, was an important reason of the biological activity enhancement of A67. According to the results of the integrated metabolome and proteome study, the increase of polyphenol content was caused by upregulation of the phenylpropanoid biosynthesis. This study expanded the understanding of active compounds and metabolic pathway of P. baumii.

Review of Advances in Bioactive Low-Molecular-Weight Compounds, Extracts, and Biology of Phellinus sensu lato Mushrooms (Agaricomycetes) from 2011 to 2017.

Various diseases such as cancer, hyperglycemia, and obesity negatively influence people's health and daily life. Mushroom species are miniature pharmaceutical factories producing hundreds of novel constituents with a long history of use in Oriental medicine. As a new therapy, they have attracted much attention owing to their potent therapeutic activity. Phellinus sensu lato (s.l.) is a well-known medicinal mushroom genus that has long been used in preventing ailments, including gastroenteric dysfunction, diarrhea, hemorrhage, and cancers, in Oriental countries, particularly in China, Japan, and Korea. Isolated compounds or complex extracts from Phellinus demonstrate specific bioactivity. Low-molecular-weight components, especially terpenoids, polyphenols, and other secondary metabolites, still exhibit biological activity. This review highlights the bioactive compounds from Phellinus s. l. mushrooms as being potent and having unlimited applications, especially the bioactive low-molecular-weight compounds, such as terpenoids, polyphenols, and their derivatives; also, pharmacological properties of the extracts and fractions from the fruiting bodies and the mycelia of Phellinus s. l., and the mechanism of the pharmacological activity have been discussed. This review summarized the two parts as mentioned above over a period of 7 years from 2011 to 2017.

Employment of ARTP to Generate Phellinus baumii (Agaricomycetes) Strain with High Flavonoids Production and Validation by Liquid Fermentation.

To obtain Phellinus baumii strain with high flavonoids yield, ARTP was employed to generate mutants of a Ph. baumii strain, which were screened for higher flavonoids content. After five rounds of screening, four mutants were identified to produce more flavonoids than the wild type strain under optimal conditions, of which A67 was the mutant with the highest flavonoids productive capacity. When cultured in shake flasks, the maximum intracellular total flavonoids production of A67 reached 0.56 g/L, 86.67% higher than the total flavonoids in CK. Antagonistic testing, RAPD, and HPLC analysis suggested that ARTP caused changes of the genetic material and metabolites in Ph. baumii. In addition, the superiority of A67 to CK was proved by liquid fermentation using unstructured kinetic models, which was performed in a 50-L fermentor. The maximum intracellular total flavonoids production and dry mycelium weight of A67 reached 0.64 g/L and 15.24 g/L, which was an increase of 88.24% and 18.23% compared with CK, respectively. This work could provide an efficient and practical strategy to obtain high flavonoids production strains and the superiority of A67 could also provide a reference to further increase flavonoids production of Ph. baumii in large-scale production mode by submerged fermentation process.

Compound of Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes), Induces Protective Autophagy in SPCA-1 Cells through AMPK Inhibition-Independent Blockade of the Akt/mTOR Pathway.

Our previous study showed that By-1, a maleimide derivative isolated from Taiwanofungus camphoratus, could induce reactive oxygen species-triggered apoptosis and G2 cell cycle arrest through a caspase-dependent pathway and also induced protective autophagy in human lung cancer SPCA-1 cells. Here, we further examined the autophagy flux and detected related proteins by Western blot analysis and fluorescence activated cell sorting, and we sought to find the exact role and underlying pathway of autophagy in SPCA-1 cells. Our results showed that By-1 treatment activated autophagy flux in SPCA-1 cells, which further confirmed that autophagy was induced by By-1 treatment in our previous study. Autophagy activator rapamycin restored cell death from By-1 treatment (21.32%) and verified that autophagy played a protective role in By-l-treated cells. Meanwhile, By-1 treatment suppressed the Akt-mammalian target of rapamycin (mTOR) pathway and the AMP-activated protein kinase (AMPK) pathway. Taken together, these findings indicate that By-1 induced protective autophagy in SPCA-1 cells through AMPK inhibition-independent blockade of the Akt/mTOR pathway.

Autophagy Inhibition Enhances SPCA-1 Cell Proliferation Inhibition Induced by By-1 from the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes).

Taiwanofungus camphoratus has been reported to have antitumor effects against various cancer cells. The aim of this study was to investigate the direct inhibitory effect of By-1 (3-isobutyl-l-methoxy-4-[4'-(3-methylbut-2-enyloxy)phenyl]-1H-pyrrole-2,5-dione), a compound from spent broth from submerged cultures of T. camphoratus, on human lung adenocarcinoma cells and to determine the molecular mechanism underlying this effect. The growth-inhibitory assay and colony formation assay showed that cell viability was significantly decreased. A By-1 concentration of 300 µmol/L caused 73.55% cell death and at a concentration of 240 µmol/L led to a 58% reduction in the number of colonies. The wound-healing assay showed that the distance of migration was 0.3 times shorter than that of untreated cells. Flow cytometry revealed that By-1 could suppress DNA synthesis, cause cell cycle arrest at the S phase, and induce apoptosis in a reactive oxygen species-dependent manner. Furthermore, the expression of caspase-3 and P53 was 4 times higher than that in untreated cells, and the antiapoptotic protein Bcl-2 was decreased 2 times compared with the protein in untreated cells. It is interesting to note that apoptosis and autophagy were both induced during treatment with By-1, and autophagy inhibition decreased cell proliferation. By-1 potently inhibited the growth of SPCA-1 cells by inducing cell cycle arrest and apoptosis. The combination of proapoptosis agents and antiautophagy agents could effectively enhance anticancer efficacy, which may be a new strategy in treating non-small cell lung cancer.

Antitumor Effect of By-1 from Spent Broth from Submerged Cultures of Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Higher Basidiomycetes), on A549 Adenocarcinoma Cells.

By-1 was obtained from spent broth from submerged cultures of Taiwanofungus camphoratus. This report evaluates the effects of By-1 on plate clone formation, wound healing, cell cycle, activated caspase-3 expression, and ROS release in A549 lung cancer cells. The result of plate clone formation assay revealed that By-1 could dramatically inhibit the viability of A549 cells in vitro. The inhibitory effect of By-1 on cell migration was tested using a wound healing assay. Proliferation rates of A549 cells were significantly inhibited following exposure to By-1 (12.5, 50, and 80 µg/mL). Flow cytometry revealed that the extracts increased, in a concentration-dependent manner, the number of cells in the G0/G1 phases of the cell cycle. The results of the caspase-3 experiment suggested that By-1 could induce A549 cells apoptosis, and this apoptosis was related to the release of reactive oxygen species by the A549 cells. All these results indicate that By-1 has potential in anti-lung cancer drug development.

Biodiesel production from soybean oil deodorizer distillate enhanced by counter-current pulsed ultrasound.

Biodiesel production from soybean oil deodorizer distillate enhanced by counter-current pulsed ultrasound was studied. Effect of static probe ultrasonic enhanced transesterification (SPUE) and counter-current probe ultrasonic enhanced transesterification (CCPUE) on the biodiesel conversion were compared. The results indicated that CCPUE was a better method for enhancing transesterification. The working conditions of CCPUE were studied by single-factor experiment design and the results showed that the optimal conditions were: initial temperature 25 °C, methanol to triglyceride molar ratio 10:1, flow rate 200 mL/min, catalyst content 1.8%, ultrasound working on-time 4 s, off-time 2 s, total working time 50 min. Under these conditions, the average biodiesel conversion of three experiments was 96.1%.