Ganoderic acid T improves the radiosensitivity of HeLa cells via converting apoptosis to necroptosis.

The use of natural substances derived from traditional Chinese medicine and natural plants as safe radiosensitizing adjuvants is a new trend for cancer radiotherapy. Ganoderma lucidum has been used as a traditional Chinese medicine with a history of more than 2000 years. Ganoderic acid T (GAT) is a typical triterpene of G. lucidum, which has strong cytotoxicity to cancer cells, but whether it has radiation sensitization effect has not been explored. In this work, we treated the HeLa cells with different concentrations of GAT before exposure to gamma-ray radiation and investigated its influence on the radiosensitivity. The cell viability, apoptosis rate, necoptosis rate, intracellular ATP level, cell cycle, the amount of H2AX and 53BP1, reactive oxygen species, and mitochondrial membrane potential were examined. Apoptotic, necroptotic, and autophagic biomarker proteins, including caspase 8, cytochrome c, caspase 3, RIPK, MLKL, P62, and LC3, were analyzed. As a result, we confirmed that with treatment of GAT, the gamma-ray radiation induced both apoptosis and necroptosis in HeLa cells, and with increase of GAT, the percentage ratio of necroptosis was increased. The involved pathways and mechanisms were also explored and discussed.

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.

Chemical Compounds and Antioxidant Activity of Volatile Oil from the White Jelly Mushroom, Tremella fuciformis (Tremellomycetes).

To fully analyze the composition of volatile oil extracted from Tremella fuciformis, hydrodistillation (HD) and solid phase microextraction (SPME) were adopted simultaneously. In both cases, the analysis was carried out using gas chromatography-mass spectrometry and the antioxidant activity of the volatile oil was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method with rutin as a positive control. Nineteen components in HD and 68 components in SPME were identified, respectively. Moreover, the oil obtained from T. fuciformis by HD indicated that aromatic compounds were a major class (93.5%), followed by the terpenes (5.7%), alkanes (0.4%), and alcohols (0.3%). Among them, butylated hydroxytoluene was the highest concentration (92.5%) of the compounds. The compounds detected by SPME were different from those of HD, and the substances with the largest content were esters (57.7%), followed by alcohols (19.0%), acids (7.0%), and aldehydes (6.3%). Only three of the same substances were detected in both of them, namely borneol, (-)-α-terpineol, and acetic acid. In the DPPH assays, strong antioxidant activity (IC50 = 0.176 mg/mL) was evident in volatile oil from T. fuciformis. Antioxidant activity was positively correlated with the concentration of volatile oil.

Dissolution of Bioactive Components from Dried Fruiting Bodies of the Culinary-Medicinal Shiitake Mushroom, Lentinus edodes (Agaricomycetes), during Cleaning, Soaking, and Cooking.

Lentinus edodes fruiting bodies are rich in active substances such as polysaccharides and eritadenine. Patients with gout, however, should avoid or severely limit their intake of foods containing large amounts of purine. In this study we quantitatively analyzed the polysaccharide and purine compounds dissolved from L. edodes fruiting bodies during cleaning, soaking, and cooking. Eritadenine, adenosine, guanosine, guanosine monophosphate, adenosine monophosphate, xanthine, and adenine dissolved from L. edodes fruiting bodies during cleaning with tap water; their dissolution rates ranged between 3.77% and 24.30%. Dissolution rates of polysaccharide and purine compounds in L. edodes fruiting bodies increased linearly with increases in the duration of soaking and cooking, and adding acetic acid or NaHCO3 in the soaking or cooking solutions significantly either inhibited or promoted their dissolution rates. On the basis of these experimental results, we offer science-based suggestions for reasonable treatment of L. edodes fruiting bodies before eating for both patients with gout and healthy people.

Effect of Culture Time on the Bioactive Components in the Fruit Bodies of Caterpillar Mushroom, Cordyceps militaris CM-H0810 (Ascomycetes).

Cordyceps militaris are widely cultivated in China for an important raw material for health foods. CM-H0810 is a C. militaris strain used in the production of C. militaris in Shanghai, the surrounding areas of Shanghai, and Guangdong province in China. We evaluated the effect of culture time on the bioactive components in the fruit bodies of C. militaris CM-H0810 to provide scientific references for production of C. militaris fruit bodies with good quality. The results showed that the polysaccharide contents increased gradually during 35-45 d, but it declined with the prolongation of culture time. The highest polysaccharide content was 3.46% at 45 d. With the prolongation of culture time the cordycepin content gradually increased; the highest cordycepin content was 3.57 µg/mg at 60 d, which increased 321% compared to that at 35 d. Contrary to cordycepin, the adenosine content declined gradually, with the highest content of 1.86 µg/mg at 35 d and the lowest content of 1.48 µg/mg at 60 d. Our study indicates that it is necessary to select suitable harvest times in view of different compounds that are desirable to obtain in high quantities.

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.

Triterpenes and Soluble Polysaccharide Changes in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), During Fruiting Growth.

We analyzed the changes in triterpenes and soluble polysaccharides in Ganoderma lucidum strain G0119 during 4 growth phases in 3 regions of the fruiting bodies using reversed-phase high-performance liquid chromatography, and we also analyzed the soluble polysaccharides using high-performance size-exclusion chroma-tography-multiple-angle laser-light scattering refractive index analysis. The strong polar triterpenes decreased while weak polar triterpenes increased during the growth cycle of G. lucidum. The highest contents of ganoderic acid B, ganoderic acid A, and ganoderenic acid B were detected in the stipe during phase II, and ganoderic acid S, ganoderic acid T, and ganoderiol B peaked in the base during phase IV. The total content of soluble polysaccharides in samples decreased after the primordium developed into a fruiting body. Two high-molecular-weight fractions were detected in the soluble polysaccharide samples: α-l,4-glucan and ß-l,3-glucan, respectively. They were primarily distributed in the pileus during phase II, and both decreased after this phase. These results led us to select a more suitable growth phase and region for harvesting to obtain extracts with higher contents of triterpenes and soluble polysaccharides.

Structural Characteristics of the Novel Polysaccharide FVPA1 from Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), Capable of Enhancing Natural Killer Cell Activity against K562 Tumor Cells.

FVPA1, a novel polysaccharide, has been isolated from fruiting bodies of the culinary-medicinal mushroom Flammulina velutipes, a historically popular, widely cultivated and consumed functional food with an attractive taste, beneficial nutraceutical properties such as antitumor and immunomodulatory effects, and a number of essential biological activities. The average molecular weight was estimated to be ~1.8 × 104 Da based on high-performance size exclusion chromatography. Sugar analyses, methylation analyses, and 1H, 13C, and 2-dimensional nuclear magnetic resonance spectroscopy revealed the following structure of the repeating units of the FVPA1 polysaccharide Identification of this structure would conceivably lead to better understanding of the nutraceutical functions of this very important edible fungus. Bioactivity tests in vitro indicated that FVPA1 could significantly enhance natural killer cell activity against K562 tumor cells.

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.

Characterization of Polysaccharides from the Fruiting Bodies of Two Species of Genus Ganoderma (Agaricomycetes) and Determination of Water-Soluble ß-D-Glucan Using High-Performance Liquid Chromatography.

Molecular weight (Mw) distributions of polysaccharides from the fruiting bodies of different Ganoderma lucidum strains and G. sinense were investigated and compared using high-pressure size exclusion chromatography/multiangle laser light scattering/refractive index analysis. Results showed that there were big differences in the Mw distributions and characteristics of polysaccharides from 2 species of Ganoderma. All tested G. lucidum materials exhibited similar polysaccharide distributions and similar characteristics for each fraction. The fraction with highest Mw (peak 1) was identified as ß-(1→3)-linked D-glucan with (1→6)-ß-D-glucopyranosyl side branches. G. sinense fruiting bodies did not include the ß-D-glucan when compared with G. lucidum. A high-pressure size exclusion chromatography method was developed and applied to determine the amount of high-Mw ß-D-glucan in G. lucidum fruiting bodies. Results indicated that there was no obvious relationship between ß-D-glucan content and the genetic similarity of G. lucidum. The strain labeled "Longzhi no. 2" was determined to possess the largest amount of ß-D-glucan: 8.2 mg/mL based on the dry weight of fruiting bodies. The ß-D-glucan content in the hot water extract of Longzhi no. 2 reached 17.05%. For the "Hunong no. 1" strain, the ß-D-glucan content in log-cultivated fruiting bodies was much higher than that in bag-cultivated ones. This method could be used to improve quality control of polysaccharides in G. lucidum.

Structural Characterization and Immunological Activities of a Novel Water-Soluble Polysaccharide from the Fruiting Bodies of Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

A water-soluble polysaccharide, designated FVPA2, was isolated from the fruiting bodies of Flammulina velutipes using DEAE Sepharose Fast Flow and gel-permeation chromatography. Its structure was elucidated by monosaccharide composition and methylation analysis, ultraviolet, Fourier transform infrared spectrometry, and nuclear magnetic resonance spectroscopy. Results showed that FVPA2 was a homogeneous heteropolysaccharide containing galactose, fucose, and mannose in a molar ratio of 5:1:1. High-performance liquid chromatography indicated its molecular weight as 3.4 × 104 Da. FVPA2 also has a repeating unit. In vitro immunomodulatory studies showed that Raw264.7 cells were stimulated to secret nitric oxide upon administration of 200-500 µg/mL FVPA2. FVPA2 also stimulated the proliferation of mouse spleen lymphocytes and B lymphocytes.

Antitumor Compounds from the Stout Camphor Mushroom Taiwanofungus camphoratus (Higher Basidiomycetes) Spent Culture Broth.

A known compound, 5-(hydroxymethyl) furan-2-carbaldehyde, and a novel compound, 3-isobutyl-1-methoxy-4-(4'-(3-methylbut-2-enyloxy)phenyl)-1H-pyrrole-2,5-dione were isolated from spent broth from submerged cultures of Taiwanofungus camphoratus. Their structures were elucidated by nuclear magnetic resonance (1H, 13C, and 2D) and mass spectra. These compounds inhibited the proliferation of K562 and HepG2 tumor cells in vitro.

Hypoglycemic Effect of Ethanol and Ethyl Acetate Extract of Phellinus baumii Fruiting Body in Streptozotocin-Induced Diabetic Mice.

We investigated hypoglycemic effect of ethanol (EtOH) and ethyl acetate extract acetate (AcOEt) extracts in streptozotocin- (STZ-) induced diabetic mice. Our data showed the maximum inhibitory effect on the fasting plasma glucose (FPG) level was detected in STZ-induced diabetic mice administered with 400 mg/kg AcOEt extract of P. baumii. A lower glycated albumin (GA) level and a higher insulin level were observed in 400 mg/kg AcOEt and EtOH extract groups. Moreover, 400 mg/kg AcOEt and EtOH extract exhibited a stronger effect on increasing size and cell number of islets. The insulin expression level of ß-cells and integrated optical density (IOD) value were significantly increased by the administration of 400 mg/kg AcOEt and EtOH extracts. Taken together, AcOEt and EtOH extracts of P. baumii fruiting body exhibited considerable hypoglycemic effect on STZ-induced diabetic mice.

Nutritional Composition of Three Domesticated Culinary-Medicinal Mushrooms: Oudemansiella sudmusida, Lentinus squarrosulus, and Tremella aurantialba.

The nutritional composition of three recently domesticated culinary-medicinal mushroom species (Oudemansiella sudmusida, Lentinus squarrosulus, and Tremella aurantialba) was evaluated for contents of protein, fiber, fat, total sugar content, amino acid, carbohydrate, and nucleotide components. The data indicated that fruiting bodies of these three mushroom species contained abundant nutritional substances. The protein contents of L. squarrosulus and O. submucida were 26.32% and 14.70%, which could be comparable to other commercially cultivated species. T. aurantialba contained 74.11% of carbohydrate, of which soluble polysaccharide was 40.55%. Oudemansiella sudmusida contained 15.95% of arabitol as the highest sugar alcohol in three mushrooms. These mushrooms also possessed distinct taste by their flavor component composition. Among them, L. squarrosulus contained 10.68% and 9.25% of monosodium glutamate-like and sweet amino acids, which were higher than the other two mushrooms. However, the nucleotide amounts of the three mushrooms were all lower than those of other commercially cultivated mushrooms. Among them, L. squarrosulus contained the highest amount of flavor nucleotides, which was 1.01‰. Results revealed that these three mushroom species are potentially suitable resources for commercial cultivation and healthy food.

Repellent constituents of essential oil of Cymbopogon distans aerial parts against two stored-product insects.

The screening for bioactive principles from several Chinese medicinal herbs showed that the essential oil of Cymbopogon distans aerial parts possessed strong repellency against the booklouse, Liposcelis bostrychophila , and the red flour beetle, Tribolium castaneum . A total of 36 components of the essential oil were identified by GC and GC-MS. trans-Geraniol (16.54%), (R)-citronellal (15.44%), (+)-citronellol (11.51%), and α-elemol (9.06%) were the main components of the essential oil followed by ß-eudesmol (5.71%) and (+)-limonene (5.05%). From the essential oil, four monoterpenes were isolated by bioassay-guided fractionation. The compounds were identified as limonene, citronellol, citronellal, and trans-geraniol. Geraniol and citronellol were strongly repellent against the booklouse, L. bostrychophila, whereas citronellal and limonene exhibited weak repellency against the booklouse. Geraniol and citronellol exhibited comparable repellency against the booklouse relative to the positive control, DEET. Moreover, geraniol and citronellol exhibited stronger repellency against the red flour beetle than DEET, whereas the two other compounds showed the same level of repellency against the red flour beetle compared with DEET.