Natural Deep Eutectic Solvent-Assisted Extraction, Structural Characterization, and Immunomodulatory Activity of Polysaccharides from Paecilomyces hepiali.

Polysaccharides, which can be affected by different preparations, play a crucial role in the biological function of Paecilomyces hepiali (PHPS) as a health food. To explore high-valued polysaccharides and reduce the negative influence of human involvement, a green tailorable deep eutectic solvent (DES) was applied to optimize the extraction of polysaccharides (PHPS-D), followed by the evaluation of the structural properties and immunomodulation by comparison with the hot-water method (PHPS-W). The results indicated that the best system for PHPS-D was a type of carboxylic acid-based DES consisting of choline chloride and succinic acid in the molar ratio of 1:3, with a 30% water content. The optimal condition was as follows: liquid-solid ratio of 50 mL/g, extraction temperature of 85 °C, and extraction time of 1.7 h. The actual PHPS-D yield was 12.78 ± 0.17%, which was obviously higher than that of PHPS-W. The structural characteristics suggested that PHPS-D contained more uronic acid (22.34 ± 1.38%) and glucose (40.3 ± 0.5%), with a higher molecular weight (3.26 × 105 g/mol) and longer radius of gyration (78.2 ± 3.6 nm), as well as extended chain conformation, compared with PHPS-W, and these results were confirmed by AFM and SEM. Immunomodulatory assays suggested that PHPS-D showed better performance than PHPS-W regarding pinocytic activity and the secretion of NO and pro-inflammatory cytokines (IL-6, TNF-α and IL-1ß) by activating the corresponding mRNA expression in RAW264.7 cells. This study showed that carboxylic acid-based DES could be a promising tailorable green system for acidic polysaccharide preparation and the valorization of P. hepiali in functional foods.

Omics data reveal the unusual asexual-fruiting nature and secondary metabolic potentials of the medicinal fungus Cordyceps cicadae.

BACKGROUND: Ascomycete Cordyceps species have been using as valued traditional Chinese medicines. Particularly, the fruiting bodies of Cordyceps cicadae (syn. Isaria cicadae) have long been utilized for the treatment of chronic kidney disease. However, the genetics and bioactive chemicals in this fungus have been largely unexplored. RESULTS: In this study, we performed comprehensive omics analyses of C. cicadae, and found that, in contrast to other Cordyceps fungi, C. cicadae produces asexual fruiting bodies with the production of conidial spores instead of the meiotic ascospores. Genome sequencing and comparative genomic analysis indicate that the protein families encoded by C. cicadae are typical of entomopathogenic fungi, including the expansion of proteases and chitinases for targeting insect hosts. Interestingly, we found that the MAT1-2 mating-type locus of the sequenced strain contains an abnormally truncated MAT1-1-1 gene. Gene deletions revealed that asexual fruiting of C. cicadae is independent of the MAT locus control. RNA-seq transcriptome data also indicate that, compared to growth in a liquid culture, the putative genes involved in mating and meiosis processes were not up-regulated during fungal fruiting, further supporting asexual reproduction in this fungus. The genome of C. cicadae encodes an array of conservative and divergent gene clusters for secondary metabolisms. Based on our analysis, the production of known carcinogenic metabolites by this fungus could be potentially precluded. However, the confirmed production of oosporein raises health concerns about the frequent consumption of fungal fruiting bodies. CONCLUSIONS: The results of this study expand our knowledge of fungal genetics that asexual fruiting can occur independent of the MAT locus control. The obtained genomic and metabolomic data will benefit future investigations of this fungus for medicinal uses.

Identification and production of a novel natural pigment, cordycepoid A, from Cordyceps bifusispora.

A novel yellow pigment, cordycepoid A, was isolated and identified from the entomogenous fungi Cordyceps bifusispora. Cordycepoid A exhibited no significant toxicity against Chinese hamster ovary (CHO) cells and mice, and showed high stability against food addictives, metal ions and heat. A liquid/solid double-phase cultural process for the production of the pigment was optimized as follows: 3 days aged liquid seed, 7.5 % inoculums, incubation temperature at 25 °C, 10 days of solid culture, and the last 5 days exposed to 200 Lx scattered light. The liquid seed medium and the solid culture medium were also optimized. Ethanol was selected as extracting solvent for its scale-up production. The optimal extracting conditions were determined as liquid/solid ratio at 20:1, extracting temperature at 40 °C, ultrasonic power at 400 W, and extracting time of 40 min.

Dynamics of a delayed rumor spreading model with discontinuous threshold control.

In this paper, we studied a delayed rumor spreading model with discontinuous threshold control. First, we studied the existence of equilibria of the subsystem. Regarding the delay as bifurcating parameter, the local asymptotic stability and Hopf bifurcation of the positive equilibrium are discussed by analyzing the corresponding characteristic equations of linearized systems. Then, we studied the existence of the sliding mode and analyzed the existence of the tangent equilibria, boundary equilibria, regular equilibria, and the stability of the pseudo-equilibrium. Finally, we provide some numerical simulations to verify the theoretical results.

Changes of serum miR-223-3p in patients with oral cancer treated with TPF regimen and the prognosis.

Changes of serum miR-223-3p in patients with oral cancer treated with TPF regimen and the prognosis were investigated. Fifty patients with oral cancer treated in the Affiliated Stomatological Hospital of Jiamusi University from March 2014 to January 2016 were enrolled in the study group, while 50 healthy subjects receiving physical examinations during the same period were enrolled in the control group. Serum expression of miR-223-3p was quantified by RT-qPCR. The diagnostic value of miR-223-3p in oral cancer was analyzed by the receiver operating characteristic (ROC) curve. Expression of miR-223-3p before and after treatment was compared. The study group was divided into the remission and the non-remission group based on the treatment outcome to analyze the predictive value of miR-223-3p. Patients were followed up for 3 years. Cox regression analysis was performed to analyze the independent prognostic factors. The relative serum miR-223-3p level was lower in the study than in the control group (P<0.001). Expression of miR-223-3p was significantly higher after treatment than before (P<0.05). Spearman's correlation analysis indicated that miR-223-3p expression before treatment gradually increased with the improvement of treatment outcome (r=0.617, P<0.001). The miR-223-3p level was markedly higher in the remission than in the non-remission group (P<0.05). The area under the ROC curve of miR-223-3p was 0.797. Multivariate Cox regression analysis demonstrated that the degree of differentiation [HR: 11.862 (95% CI: 2.730-51.547)] and miR-223-3p [HR: 3.489 (95% CI: 1.447-8.413)] were independent prognostic factors. The 3-year survival of patients with high differentiation and high miR-223-3p expression was significantly higher than that of patients with poor differentiation and low miR-223-3p expression (P<0.05). In conclusion, miR-223-3p expression is low in oral cancer, and it shows potential for predicting the efficacy and prognosis of patients with oral squamous cell carcinoma (OSCC) after TPF regimen.

[Anti-tumor compounds from submerger fermentation products of a Paecilomyces militaris strain].

OBJECTIVE: The aim was to identify the antitumor compounds of the fermentation broth of Paecilomyces militaris (strain RCEF0927). METHODS: Anti-tumor activity was tested with a resazurin cytotoxicity assay model with Chinese hamster ovary (CHO) cells. Bioactive compounds were analysed with combined method of High Performance Liquid Chromatography (HPLC), High Resolution Mass Spectrometer (HRMS) and antitumor assay. RESULTS: Antitumor test showed that the fermentation broth had strong cytotoxicity against CHO cells. Extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD- HRMS-Cytotoxicity assay revealed that the molecular formula of the active compounds in the extract were possibly C10 H13 N5 O3, C22 H22 O9, C23 H24 O10, C22 H22 O10, C15 H10 O4, C15 H10O, C36 H70 O11, C36 H68 O12, C38 H74 O11, C40 H76 O13 and C44 H82 O14. CONCLUSION: The possible structures of the bioactive compounds were deduced with bioassay, HRMS, Uv and database inquiry as cordycepin, daizein, genistein 7,4'-dihydroxyisoflavone-7-O-(4"-O-methyl)-b-D-glucopyranoside, 7, 4'-dihydroxy-6- methoxyisoflavone-7-O-( 4"-O-methyl)-b-D-glucopyranoside, 5, 7, 4'-trihydroxy-isoflavone-7-O-(4"-O-methyl)-b-D-glucopyranoside, polyoxyethylene ether oleate, dehydro products of polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxypropylene or polyoxybutene contained derivants of polyoxyethylene sorbitan fatty acid ester. Cytotoxicity of the compounds was revealed for the first time.

Potential therapeutic effects of Cordyceps cicadae and Paecilomyces cicadae on adenine-induced chronic renal failure in rats and their phytochemical analysis.

BACKGROUND: Natural Cordyceps cicadae (C. cicadae) has been utilized extensively in traditional Chinese medicine to treat chronic renal diseases, heart palpitations, infantile convulsions, and dizziness. However, given its slow growth and immoderate exploitation, C. cicadae resources have been severely depleted. By contrast, Paecilomyces cicadae (P. cicadae), as the anamorph stage of C. cicadae, is easy to cultivate, and this kind of cultivated P. cicadae has good and controllable quality. PURPOSE: This study aimed to compare the therapeutic effects of C. cicadae and P. cicadae on adenine-induced chronic renal failure (CRF) rats. In accordance with the aforementioned studies, our work subsequently analyzed the intrinsic relationships between the efficacy and pharmacodynamic substances of C. cicadae and P. cicadae to conclude whether or not P. cicadae could be used as an alternative to C. cicadae in treating CRF. METHODS: Rats were administered with C. cicadae (1.0 g/kg) or P. cicadae (1.0 g/kg) by gavage for 4 weeks. Furthermore, we applied Fourier transform infrared spectroscopy, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and ultraviolet spectrophotometry to comprehensively detect and analyze the chemical constituent differences from ten batches each of C. cicadae and P. cicadae. RESULTS: This study revealed that both C. cicadae and P. cicadae exerted obvious therapeutic effects on CRF and were more consistent with their chemical compositions. CONCLUSION: P. cicadae can be used as an alternative to C. cicadae for treating CRF to cater to market demands.

Bioactive compounds from Paecilomyces tenuipes regulating the function of the hypothalamo-hypophyseal system axis in chronic unpredictable stress rats.

BACKGROUND: A bioactive compound from Paecilomyces tenuipes (BCPT) has an inhibitory effect on monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in vitro and in vivo, which indicates BCPT may be a potential antidepressant. In this study we aimed to study the antidepressant effects of BCPT in the chronic unpredictable stress (CUS) model in rats and explore underlying mechanisms in the hypothalamic-pituitary-adrenal (HPA) axis. METHODS: The antidepressant effects of BCPT were studied in the chronic unpredictable stress model in rats. Animals were housed isolated, except the control group. Rats were exposed daily to different random stressors from day 1 to 21. Awarding response was detected by calculating the 24-hour consumption of sucrose water. Cortisol (CORT) and adrenocorticotropic hormone (ATCH) contents in serum and arginine vasopressin (AVP) contents in the pituitary body were detected by radio immunoassays. Total RNA of hippocampus or hypothalamus was extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the measurement of corticotrophin releasing hormone (CRH) mRNA or mineralocorticoid receptor (MR) mRNA and glucocorticoid receptor (GR) mRNA levels. Statistical analyses were performed using one way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) test. RESULTS: Chronic unpredictable stress resulted in reduction of sensitivity to reward and abnormality in the HPA axis in the animal model. BCPT improved the reward reaction as measured by increasing sucrose consumption, remarkably reduced serum CORT and ACTH levels and the AVP content in the pituitary body in the CUS-treated rats, decreased the expression of CRH mRNA, enhanced the expression of hippocampus MR mRNA, GR mRNA and decreased the ratio of MR/GR. CONCLUSIONS: BCPT has potentially antidepressant-like activity and normalized the HPA axis hyperactivity in a CUS model of depression in rats. This may be an important mechanism of its antidepressant effect.

Analysis of radical scavenging active components in the fermented mycelia of Ophiocordyceps formosana.

The rates of 1,1-diphenyl-2-picryhydrazyl (DPPH)-free radical scavenging and hydroxyl radical eliminating were employed as indexes to qualitatively and quantitatively analyse the radical scavenging activity of the extracts with different solvents from fermented mycelia of Hirsutella huangshanensis RCEF0868, the anamorph of Ophiocordyceps formosana (FMOF). The results showed that both the aqueous extract and the methanol extract had significant radical scavenging activity. The half maximal inhibitory concentrations (IC50) of the aqueous extract on DPPH-free radical and hydroxyl radical are 0.85 and 1.37 mg/mL, respectively, while the IC50 of methanol extract is 1.23 and 2.91 mg/mL, respectively. The combined liquid chromatography-diode array detector-high resolution mass spectrometer(HRMS) analysis and activity determination revealed that the molecular formulas of three radical scavengers in aqueous extract were C6H14O6(1), C14H17N5O8(2) and C20H33N5O9(3). In addition to the compound 1, the methanol extract had another three radical scavengers as C30H22O11(4), C30H22O10(5) and C30H18O10(6). All the six kinds of components exhibited the DPPH radical scavenging activity, and compounds 1, 4 and 5 also showed the hydroxyl radical eliminating activity at the same time. By querying the database of natural products and reference to the relevant reports, compound 3 may be a new entity. The others were respectively cordycepic acid, succinoadenosine, 4α-oxyrugulosin, rugulosin and skyrin and, among them, succinoadenosine and 4α-oxyrugulosin were found in entomogenous fungi for the first time.

Determination and comparative analysis of 13 nucleosides and nucleobases in natural fruiting body of Ophiocordyceps sinensis and its substitutes.

Nucleosides and nucleobases are one of the most important indicators of quality control. A sensitive and reliable high performance liquid chromatography-ultraviolet method was applied to analyse 13 nucleosides and nucleobases simultaneously in 15 batches of nine Ophiocordyceps species and its allies in China. Principal component analysis (PCA) and cluster analysis were conducted by SPSS 22.0 software (IBM Corp., Armonk, NY, USA). The 15 samples of Cordyceps were differentiated successfully based on their nucleoside and nucleobase content. Total nucleosides content in mycelium was significantly higher than that in the natural fruiting bodies of Ophiocordyceps sinensis (NFOS). Five nucleosides or nucleobases - adenine (A), guanosine (Gu), uracil (U), uridine (Ur) and guanine (G) - were the major components contributed to the total variance according to PCA. The profiles of the 13 tested nucleosides and nucleobases (including adenosine, cytidine, guanosine, inosine, thymidine, uridine, cordycepin, adenine, cytosine, guanine, thymine, uracil and hypoxanthine) can discriminate different samples and can be candidate indicators applied for the quality control of Ophiocordyceps and its allies.

Identification and determination of myriocin in Isaria cicadae and its allies by LTQ-Orbitrap-HRMS.

A hybrid linear ion trap-quadrupole-Orbitrap high-resolution mass spectrometry (LTQ-Orbitrap-HRMS) was used to qualitatively and quantitatively analyse the myriocin in Isaria cicadae and its allies. The samples were prepared with 95% methanol for 30 min by ultrasonic-assisted extraction. The target compound was purified by ODS solid-phase extraction (SPE) column. The enriched samples were identified by mass spectrometry. The results showed that the contents of myriocin in both wild and artificial Isaria cicadae were below the detection limit, while a strain of Ophiocordyceps longissima and Cordyceps cicadae Shing (Dujiaolong), both closely related to the Isaria cicadae, and its asexual mycelia are rich in myriocin. It suggests that it may be wrong to consider C. cicadae as I. cicadae's teleomorph in Genbank or Mycobank in many published reports based on chemical classification, and the species rich in myriocin is probably not Isaria cicadae.

New tyrosinase inhibitors from Paecilomyces gunnii.

Through screening 50 strains of entomopathogenic fungi and rescreening of 7 strains of Paecilomyces gunnii, a methanol extract of liquid-cultivated mycelia of P. gunnii was found to have the strongest tyrosinase inhibitory activity. Preparative high-speed counter-current chromatography (HSCCC) guided by high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-high-resolution mass spectrometry (HRMS) was employed for the isolation and purification of the active components, and three new compounds with half inhibition concentration (IC50) of 0.11, 0.17, and 0.14 mM against diphenolase were obtained from the extract, respectively. Their chemical structures were identified by HRMS, one- and two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy as paecilomycones A, B, and C. Structure and activity studies showed that the tyrosinase inhibition activities are positively related to the number of hydroxyl groups on the paecilomycones.

Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana.

A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies and infected insects. This suggests that using cultured mycelia to substitute a natural Cordyceps may result in poisoning. A combined method of HPLC-PAD-HRMS and cytotoxic analysis revealed that the most toxic compound (Compound 1) was found mainly in the cultured mycelia and also a small amount in the infected insect body of the Cordyceps, but not in the fruiting body. The second toxic compound (Compound 2) was found in all structures of Cordyceps and in cultured mycelia. Different contents of the toxic compounds resulted in the different cytotoxicity of the extracts. Compound 1 and Compound 2 were prepared with preparative HPLC as yellow and orange powders, respectively. Cytotoxic tests showed that the median lethal dose (LD50) against CHO cells of Compound 1 was 18.3 ± 0.2 and 103.7 ± 5.9 µg/mL for Compound 2. Compound 1 and Compound 2 were identified as rugulosin and skyrin by HRMS, UV and NMR data. The two compounds were never previously isolated from the genera Cordyceps and Hirsutella and their cytotoxicity against CHO cells was also reported for the first time.

Anti-tumor Effects of Exo- and Endo-biopolymers Produced from Submerged Cultures of Three Different Mushrooms.

The anti-tumor effects of exo- (EX) and endo-biopolymers (EN) produced from submerged mycelial cultures of Ganoderma applanatum (GA), Collybia confluens (CC), and Pleurotus eryngii (PE) were studied using Sarcoma 180 bearing mice. Solid tumor growth was inhibited most effectively when 40 mg/kg body weight (BW) of GA-EX or PE-EN was administered to the intraperitoneal (i.p.) cavity of BALB/c mice. The spleen and liver indexes were increased in mice following i.p. administration of GA-EX and PE-EN fractions. GA-EX and PE-EN reduced the tumor formation by 30.7% and 29.4%, respectively. GA-EX and PE-EN increased the natural killer (NK) cell activity of splenocytes by 41.3% and 28.9%, respectively.