Development of an efficient method for separation and purification of cordycepin from liquid fermentation of Cordyceps militaris and analysis of cordycepin antitumor activity.

Cordycepin (3 '-deoxyadenosine) is the main active component of Cordyceps militaris, which is a chemical marker for quality detection of Cordyceps militaris and has important medicinal development value. Existing methods for obtaining cordycepin are complex and costly. In this study, an economical and simple method for separation and purification of cordycepin from Cordyceps militaris fermentation liquid through physical crystallization was explored. First, lyophilized powdered fermentation liquid (LPFL) and pure methanol (1 g/100 mL, w/v) were mixed, and then repeatedly dissolved and crystallized until the precipitation was white. Purified product was obtained by freeze-drying the precipitate. The substance was determined to be cordycepin by high performance liquid chromatography, mass spectrometry and infrared spectroscopy, and the purity was 94.26%. Compared with the existing methods, this method is simple and low cost. In addition, the functional activity of cordycepin was determined by in vitro test. The results exhibited that cordycepin caused death and morphological changes in human colon cancer Caco-2 cells, and significantly inhibited the proliferation of Caco-2 cells, with a half-maximal inhibitory concentration (IC50) of 107.2 µg/mL. Cordycepin could induce early apoptosis of Caco-2 and caused cell cycle arrest in the G2 phase. Caco-2 cell apoptosis and cell cycle arrest showed dose dependence to cordycepin over a certain range. These results improved cordycepin purification method, provided insights into the mechanism of cordycepin in cancer inhibition, and would provide important reference for further development and clinical application of cordycepin.

Luteimonas galliterrae sp. nov., isolated from poultry farm soil.

Strain SX5T was isolated from the soil of a poultry farm in Shanxi Province, PR China. The isolate was a Gram-stain-negative, rod-shaped, non-flagellated, and yellow bacterium. Growth occurred at 20-37 °C (optimum, 28 °C), pH 6.0-10.0 (optimum, pH 8.0) and 0-1 % NaCl (optimum, 0 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SX5T was related to members of the genus Luteimonas, and close to Luteimonas gilva H23T (97.9 %), Luteimonas cucumeris Y4T (97.9 %), Luteimonas aquatica RIB1-20T (96.8 %), Luteimonas notoginsengisoli SYP-B804T (96.4 %) and Luteimonas panaciterrae Gsoil 068T (96.1 %). The major cellular fatty acids of strain SX5T were iso-C16 : 0, iso-C17 : 1 ω9c, iso-C15 : 0 and iso-C11 : 0 3OH. The sole isoprenoid quinone was ubiquinone Q-8, and the major polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Genome analyses revealed that strain SX5T had a genome size of 3.6 Mbp with a G+C content of 65.7 mol% and contained abundant carbohydrate-active enzyme genes and three putative distinct biosynthetic gene clusters, suggesting that it may have great potential to degrade and utilize complex biological organic matter and produce special secondary metabolites. Comparative genomic analyses clearly separated strain SX5T from the known species of the genus Luteimonas based on average nucleotide identity and digital DNA-DNA hybridization values below the thresholds for species delineation. Based on its phenotypic, genotypic properties and phylogenetic inference, strain SX5T represents a novel species in the genus Luteimonas, for which the name Luteimonas galliterrae sp. nov. is proposed. The type strain is SX5T (=GDMCC 1.2162T=KCTC 82443T=JCM 34401T).

Roseococcus pinisoli sp. nov., lacking pufL and pufM bacteriochlorophyll a: synthesizing genes.

A Gram-stain-negative, cocci-to-oval-shaped bacterial strain, designated XZZS9T, was isolated from the rhizosphere soil of Pinus sylvestris var. mongolica and characterized taxonomically using a polyphasic approach. Growth occurred at 20-35 °C (optimum, 28 °C), pH 6.0-11.0 (optimum, pH 7.0), and in 0-1% NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain XZZS9T was related to members of the genus Roseococcus, with the highest sequence identity to Roseococcus microcysteis NIBR12T (96.9%). The major cellular fatty acids (> 5% of the total) were C18:1 ω7c and C19:0 cyclo ω8c. The major isoprenoid quinone was Q-9 and the polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an unidentified glycophospholipid, and an unidentified phospholipid. Genome sequencing revealed that had a genome size of 4.79 Mbp with a G + C content of 69.5%. Comparative genomic analyses clearly separated strain XZZS9T from the known species of the genus Roseococcus based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values below the thresholds for species delineation. Genome annotations did not find pufL and pufM genes in strain XZZS9T, suggesting a possible lack of photosynthetic reaction. Based on genotypic and phenotypic characteristics, strain XZZS9T represents a novel species of the genus Roseococcus, for which we propose the name Roseococcus pinisoli sp. nov. The type strain is XZZS9T (= KCTC 82435T = JCM 34402T = GDMCC 1.2158T).

A non-symbiotic novel species, Rhizobium populisoli sp. nov., isolated from rhizosphere soil of Populus popularis.

Strain XQZ8T, isolated from the rhizosphere soil of a Populus popularis plant in China, was characterized using a polyphasic taxonomic approach. Cells were Gram-negative, aerobic, non-spore-forming, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain XQZ8T was related to members of the genus Rhizobium and had the highest 16S rRNA gene sequence similarity to Rhizobium smilacinae PTYR-5T (96.6%). The average nucleotide identity and digital DNA-DNA hybridization value between strain XQZ8T and R. smilacinae PTYR-5T were 77.5% and 21.4%, respectively. TYGS whole-genome-based taxonomic and multi-locus sequence analyses of three concatenated housekeeping genes (atpD-recA-glnII) further indicated that strain XQZ8T was a new member of the genus Rhizobium. The major cellular fatty acids included summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 2 (C12:0 aldehyde/unknown 10.928), C16:0, and C19:0 cyclo ω8c. The major respiratory quinones were Q-9 and Q-10. The polar lipids were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylmonomethylethanolamine, an unidentified glycophospholipid, and three unidentified lipids. The genomic DNA G + C content of the strain was 60.1 mol%. Based on the phylogenetic, phenotypic, and genotypic characteristics, strain XQZ8T represents a novel species of the genus Rhizobium, for which the name Rhizobium populisoli sp. nov. is proposed. The type strain is XQZ8T (= JCM 34442T = GDMCC 1.2201T).

In Vitro Antibacterial and Antitumor Activity of Total Triterpenoids from a Medicinal Mushroom Sanghuangporus sanghuang (Agaricomycetes) in Liquid Fermentation Culture.

We analyzed the biological activity and composition of total triterpenoids extracted from the liquid fermentation culture of the medicinal mushroom Sanghuangporus sanghuang. S. sanghuang total triterpenoid extract (STTE) had the strongest inhibitory effect on Staphylococcus aureus, and STTE at 2-fold the minimum inhibitory concentration had a significant effect on the growth of four types of bacteria and caused marked nucleic acid leakage. Scanning electron microscopy confirmed that STTE destroyed bacterial cell walls. STTE also significantly inhibited the proliferation of Caco-2 cells and disrupted their morphology. Flow cytometry revealed that STTE induced cell cycle arrest and induced early and late apoptosis of Caco-2 cells. Infrared spectroscopy, and gas chromatography-mass spectrometry resulted in the detection of a variety of triterpenoids (e.g., pentacyclic triterpenoid, lanolinane triterpenoid, and trans-squalene) as well as other small molecules (e.g., esters, acids, and aldehydes). Our findings indicate that STTE has antibacterial and antitumor activity and will enable screening of novel fungal-derived anticancer and antibacterial compounds.

Letter: Electrospray ionization mass spectral characteristics and fragmentation mechanisms of triterpenoids in Fomes officinalis.

The electrospray ionization tandem mass spectrometric characteristics and fragmentation mechanisms of 12 triterpenoid compounds from Fomes officinalis (F. officinalis) and their analogs were investigated. The compounds could be classified into three types depending on their chemical structures. All of the compounds gave [M-H](-) and [2M-H](-) ions by electrospray negative ionization mode. In addition, the members of three isomeric groups of the analogs with the same elemental composition can be distinguished by tandem mass spectra of protonated molecules and of significant fragmention. The above fragmentations were reported for the first time and were implemented for the analysis of triterpenoids in F. officinalis.