[Cordyceps sinensis protects HK2 cells from ischemia-reperfusion injury through Sirt1 pathway].

OBJECTIVE: To investigate the effects of Cordyceps sinensis (CS) on cellular apoptosis and Sirt1 expression in HK2 cells followed by ischemia-reperfusion (I/R).
 Methods: HK2 cells were incubated with different concentrations of CS (10, 20, 40, 80, 160, 320 mg/L) for 24 hours, and the optimal concentration of CS was selected by measuring cell proliferation. The confluent HK2 cells were incubated with 0.01 µmol/L antimycin A for 2 hours to induce ischemia in vitro, and then the reperfusion was achieved by incubating cells with glucose-replete complete growth medium for 24 hours. HK2 cells were divided into 4 groups: a control group, an I/R group, an I/R+CS (160 mg/L) group, and an I/R+CS (160 mg/L)+Sirtinol (25 µmol/L) group. Twenty-four hours later, total RNA and protein were collected. The cell proliferation was evaluated by MTT assay; the mRNA and protein expression of Sirt1 and the cleaved caspase-3 were measured by qRT-PCR and Western blot, respectively. The cellular apoptosis rate was determined by Annexin V-FITC/PI double staining and flow cytometry.
 Results: Certain concentrations (10-160 mg/L) of CS did not show effect on the proliferation of HK2 cells (P>0.05), while 320 mg/L of CS inhibited cell proliferation significantly (P<0.01); compared with the control group, the mRNA and protein expressions of Sirt1 and the cleaved caspase-3 in the I/R group were up-regulated (P<0.01) and the apoptosis rate was extremely high; compared with the I/R group, CS significantly up-regulated Sirt1 mRNA and protein expression (P<0.01) while down-regulated cleaved caspase-3 mRNA and protein levels (P<0.01), and reduced apoptosis rate (P<0.05). The effects of CS were blocked in the presence of sirtinol, an inhibitor of CS.
 Conclusion: CS protects HK2 cells from I/R injury through activation of Sirt1 pathway.

Hypouricemic actions of exopolysaccharide produced by Cordyceps militaris in potassium oxonate-induced hyperuricemic mice.

The hypouricemic actions of exopolysaccharide produced by Cordyceps militaris (EPCM) in potassium oxonate-induced hyperuricemia in mice were examined. Hyperuricemic mice were administered intragastrically with EPCM (200, 400 and 800 mg/kg body weight) or allopurinol (5 mg/kg body weight) once daily. Serum uric acid, blood urea nitrogen and liver xanthine oxidase (XOD) activities of each treatment were measured after administration for 7 days. EPCM showed dose-dependent uric acid-lowering actions. EPCM at a dose of 400 mg/kg body weight and allopurinol showed the same effect in serum uric acid, blood urea nitrogen and liver XOD activities in hyperuricemic mice. An increase in liver XOD activities was observed in hyperuricemic mice due to administration of EPCM at a dose of 200 mg/kg body weight. EPCM at a dose of 800 mg/kg body weight did not show significant effects on serum uric acid and XOD activities. We conclude that EPCM has a hypouricemic effect caused by decreases in urate production and the inhibition of XOD activities in hyperuricemic mice, and this natural product exhibited more potential efficacy than allopurinol in renal protection.

Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene.

Like other filamentous fungi, the medicinal ascomycete Cordyceps militaris frequently degenerates during continuous maintenance in culture by showing loss of the ability to reproduce sexually or asexually. Degeneration of fungal cultures has been related with cellular accumulation of reactive oxygen species (ROS). In this study, an antioxidant glutathione peroxidase (Gpx) gene from Aspergillus nidulans was engineered into two C. militaris strains, i.e., the Cm01 strain which can fruit normally and the Cm04 strain which has lost the ability to form fruiting bodies on different media through subculturing. The results showed that the mitotically stable mutants had higher Gpx activities and stronger capacity to scavenge cellular ROS than their parental strains. Most significantly, the fruiting ability of Cm04 strain was restored by overexpression of the antioxidant enzyme. However, after being successively transferred for up to ten generations, two of three Cm04 mutants again lost the ability to fruit on insect pupae while Cm01 transformants remained fertile. This study confirms the relationship between fungal culture degeneration and cellular ROS accumulation. Our results indicate that genetic engineering with an antioxidant gene can be an effective way to reverse fungal degeneration during subculturing.

Effects of Tween 80 and pH on mycelial pellets and exopolysaccharide production in liquid culture of a medicinal fungus.

This study investigated the effects of surfactant additives and medium pH on mycelial morphology and exopolysaccharide (EPS) production in liquid culture of a valuable medicinal fungus Cordyceps sinensis Cs-HK1. In the medium containing 20 g l⁻¹ glucose and 6 g l⁻¹ peptone as the sole nitrogen source, the Cs-HK1 fungal mycelia formed smooth and spherical pellets about 1.8-mm mean diameter. The mycelial pellets became less uniform at pH (4.0-5.0) lower than the optimum (pH 6.0) or turned to filamentous form at higher pH (8-9). Surfactants added to the medium inhibited pellet formation, resulting in smaller and looser pellets. Tween 80 exhibited a remarkable promoting effect on EPS production, increasing the EPS yield more than twofold at 1.5% (w/v), which was most probably attributed to the stimulation of EPS biosynthesis and release from the fungal cells by Tween 80.

In vitro and in planta compatibility of insecticides and the endophytic entomopathogen, Lecanicillium lecanii.

In an attempt to clarify the potential role of endophytic fungi in integrated pest management, the compatibility of an endophytic isolate of Lecanicillium lecanii (Zimmermann) Gams & Zare (Hyphomycetes) with nine insecticides used against Aphis gossypii Glover (Homoptera : Aphididae) was examined both in vitro over 14 days and in planta. In the laboratory, most insecticides partially or completely inhibited the germination of conidia and growth of hyphae in nutrient-rich conditions. Endosulfan completely inhibited the germination of conidia and hyphal growth. In contrast, all insecticides were compatible with L. lecanii in planta, and the fungus was readily recovered from inoculated, colonized leaves. These data support the hypothesis that endophytic L. lecanii will be unaffected by insecticides and could be integrated in the management of pests in cotton.

The transcriptome analysis on urea response mechanism in the process of ergosterol synthesis by Cordyceps cicadae.

Nitrogen source is required for the growth of Cordyceps cicadae and involved in the regulation of metabolite synthesis. In order to further investigate the regulatory effects of nitrogen sources on the ergosterol synthesis by C. cicadae. We first confirmed that urea could significantly increase the ergosterol synthesis. The transcriptome analysis showed that compared with biomass cultured in the control fermentation medium (CFM), 1340 differentially expressed genes (DEGs) were obtained by Gene Ontology (GO) annotation, and 312 DEGs were obtained by Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation from the biomass cultured in CFM + CO(NH2)2. Urea up-regulated D-3-phosphoglycerate dehydrogenase gene transcription level and down-regulated enolase and L-serine/L-threonine ammonialyase gene transcription level, increased serine synthesis, allosterically activate pyruvate kinase, to promote the synthesis of pyruvate and CH3CO ~ SCOA, the primer of ergosterol; Urea increase the genes transcription related with ergosterol synthesis by up-regulating the steroid regulatory element binding protein gene transcription levels. The transcriptome results were provided by those of qRT-PCR. Collectively, our finding provided valuable insights into the regulatory effect of nitrogen source on the ergosterol synthesis by C. cicadae.

Fungal tolerance to Congo red, a cell wall integrity stress, as a promising indicator of ecological niche.

Differential sensitivities to the cell wall stress caused by Congo red (CR) have been observed in many fungal species. In this study, the tolerances and sensitivities to CR was studied with an assorted collection of fungal species from three phylogenetic classes: Sordariomycetes, Dothideomycetes, and Eurotiomycetes, three orders, and eight families. These grouped into different ecological niches, such as insect pathogens, plant pathogens, saprotrophs, and mycoparasitics. The saprotroph Aspergillus niger and the mycoparasite Trichoderma atroviride stood out as the most resistant species to cell wall stress caused by CR, followed by the plant pathogenic fungi, a mycoparasite, and other saprotrophs. The insect pathogens had low tolerance to CR. The insect pathogens Metarhizium acridum and Cordyceps fumosorosea were the most sensitive to CR. In conclusion, Congo red tolerance may reflect ecological niche, accordingly, the tolerances of the fungal species to Congo red were closely aligned with their ecology.

Chemical structure and mechanism of polysaccharide on Pb2+ tolerance of Cordyceps militaris after Pb2+ domestication.

In this study, the purified polysaccharide (DCP-I) was extracted from Cordyceps militaris domesticated with Pb2+. After that, the structural feature and mechanism of lead resistance of DCP-I were investigated using novel approaches. The results showed that the average molecular weight of DCP-I was 1.206 × 103 kDa and mainly consist of Rhamnose, Galactose, Glucose, Galacturonic acid and Glucuronic acid in a molar ratio of 0.130:47.687:40.784:1.795:0.48. Besides, the main chain of DCP-I was composed by →6)-Galp-(1→, →4)-Glcp-(1→ and →1,4)-Glcp-(6→, while the side chain was →1)-Rhaf-(2→ and D-Glcp-(1→, and the DCP-I contained Alacturonic acid and Glucuronic acid. In addition, the result of Congo red test showed that DCP-I did not exist triple-helical structures. SEM, EDX and XPS analyses results showed that the functional groups of DCP-I related to C, H and O (-OH, -COOH and -C=O) could combined with Pb2+effectively. The adsorption processes were described by the Pseudo-second-order kinetic model (R2 = 0.9978) and Langmuir isotherm (R2 = 0.9979) for Pb2+ indicating that adsorption process of DCP-I to Pb2+ was a kind of single molecular layer chemical adsorption.

Protective effects of Corbrin Capsule against permanent cerebral ischemia in mice.

Corbrin Capsule is a traditional Chinese patent medicine with the main component of fermentative cordyceps fungus powder (Cs-C-Q80). The indications of Corbrin Capsule include chronic renal insufficiency, chronic obstructive pulmonary (COPD), pulmonary fibrosis, and chronic bronchitis. However, the effects of Corbrin Capsule on acute cerebral ischemia are still unclear. The objective of this study was to explore the preventive effect of Corbrin Capsule in permanent and transient middle cerebral artery occlusion (MACO) mice model. Male C57BL/6 mice were given Corbrin of 0.04, 0.2 and 1 mg/kg by gavage once a day for 3, 7 or 14 days and then subjected to pMCAO or tMCAO. Infarct volumes, neurological deficit score, ATP concentration, SOD activity and MDA content were assessed. Results showed that prolonged pretreatment with Corbrin (1.0 mg/kg) to 7 days or more effectively ameliorated brain infarct and neurological scores in pMCAO mice. Shorter (3 days) or without pretreatment of Corbrin was invalid, suggesting a pretreatment time window. The ATP concentration was significantly increased with effective Corbrin pretreatments in ischemic brains, while the content of MDA sharply decreased in Corbrin groups. In tMCAO mice, Corbrin showed no neuroprotection even with pretreatment. In conclusion, long-term pre-administration of Corbrin Capsule is necessary for its anti-cerebral ischemic effects, and the underlying mechanisms might be associated with increase of ATP concentration and the anti-inflammatory effects in ischemic brain tissue.

An Efficient Strategy for Enhancement of Bioactive Compounds in the Fruit Body of Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), by Spraying Biotic Elicitors.

Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.

Increasing of the Contain of Carotenoids in Caterpillar Mushroom, Cordyceps militaris (Ascomycetes) by Using the Fungal Elicitors Cultivation.

As a natural pigment, cordycepic carotenoids have many bioactive functions, such as antiinflammation, anticancer, and antioxidation. In addition, the good coloring of this hydrophilic pigment enables it to have wide application in the food industry. This study investigated five species of fungal elicitors, namely, Rhodotorula glutinis, Saccharomyces cerevisiae, Monascus ruber, Blakeslea trispora, and Flammulina velutipes, to evaluate their effects on carotenoid accumulation in Cordyceps militaris. Results showed that all fungal elicitors, except Rh. glutinis, have no positive effect on the biosynthesis of cordycepic carotenoids. The Rh. glutinis elicitor remarkably stimulated the accumulation of carotenoids with a 13.72% increase compared with the control. Subsequently, the entire Rh. glutinis elicitor (part NHK) was divided into three parts, namely, exopolysaccharide (EPS) (part E), mixture of EPS and protein (part PE), and other components (part O), to analyze their effects on carotenoid accumulations. Results showed that part O may be the effective component that remarkably stimulates the biosynthesis of carotenoids with a 26% increase compared with the control. This research demonstrated that Rh. glutinis elicitor can effectively increase the content of natural carotenoids in C. militaris, and provided an important reference for the development and utilization of carotenoid industrialization.

Developing a Novel Two-Stage Process for Carotenoid Production by Cordyceps militaris (Ascomycetes).

Natural carotenoids are attracting increasing interest, but their widespread use is limited because of poor production. Cordyceps militaris, a traditional Chinese mushroom, contains a large amount of carotenoids, and this study aimed to increase carotenoid production by C. militaris by optimizing a liquid-state cultivation system. We developed and optimized a novel 2-stage process, including cultivation under shaking in darkness and under static irradiation on a flat panel, using response surface methodology, and we compared this process to common shake-flask cultivation. In addition, we examined the effects of different inducers (chitosan, peanut oil, tomato juice, yeast, and metal ions) on carotenoid production. Results showed that under optimal conditions (4 days of shaking in darkness, 10 days of static irradiation with a 100-mL flat panel volume), a maximum of 1217.5 ± 115.9 µg/g carotenoids were produced; only 662.9 µg/g were produced by common shake-flask cultivation. Only a large amount of chitosan (8 mg) could significantly increase carotenoid content; some of the other inducers showed inhibitory effects. This study demonstrated that this 2-stage process could effectively increase the natural carotenoid content in C. militaris, making it a potential source for commercial exploitation.

Selenium Biofortification and Antioxidant Activity in Cordyceps militaris Supplied with Selenate, Selenite, or Selenomethionine.

Selenium (Se) is an essential trace element with multiple functions that may help mitigate adverse health conditions. Cordyceps militaris is an edible mushroom with medicinal properties. The experiment was conducted under artificial cultivation, with five Se concentrations (0, 5, 10, 20, and 40 µg g-1) and three forms of Se (selenate, selenite, and selenomethionine). C. militaris can absorb inorganic from the substrate and convert it to organic Se compounds (selenocystine, selenomethionine, and an unknown species) in fruiting bodies. Compared with the control treatment, Se applications (40 µg g-1 selenate and selenite) significantly increased the Se concentration in fruiting bodies by 130.9 and 128.1 µg g-1, respectively. The biofortification with selenate and selenite did not affect fruiting body production, in some case, but did enhance the biological efficiency. Moreover, the abundance of cordycepin and adenosine increased, while the amino acid contents remained relatively stable. Meanwhile, Se-biofortified C. militaris showed effective antioxidant activities. These results suggest that Se-biofortified C. militaris fruiting bodies may enhance human and animal health when it was included as part of a healthy diet or used as Se supplements.

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation.

AIMS: To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called 'ion beam'. METHODS AND RESULTS: Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8-azaadenine- and 28 classes of 8-azaaguanine-resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8-azaadenine-resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8-azaaguanine-resistant mutants, it was shown that mutant no. G81-3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3.1 and 1.8 g l(-1) on 21-day culture for mutant no. G81-3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. CONCLUSIONS: The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large-scale production of cordycepin.

[Investigations on cordycepin production by solid culture of Cordyceps militaris].

An efficient method to produce cordycepin by solid culture using Cordyceps militaris was investigated in this study. Firstly, the changes of cordycepin during various growing periods of solid culture using 5 strains of C. militaris were detected, the best strain and optimal growing period for cordycepin production were determined. Then, by experiments of quadratic rotation-orthogonal combination design and orthogonal design, the medium composition and growth conditions for high yield of cordycepin were optimized. With the optimized method to produce cordycepin, the content of cordycepin in the medium was increased to 0.60%, which was nearly 2 times higher than the highest yield reported.

Optimal conditions for cordycepin production in surface liquid-cultured Cordyceps militaris treated with porcine liver extracts for suppression of oral cancer.

Cordycepin is one of the most crucial bioactive compounds produced by Cordyceps militaris and has exhibited antitumor activity in various cancers. However, industrial production of large amounts of cordycepin is difficult. The porcine liver is abundant in proteins, vitamins, and adenosine, and these ingredients may increase cordycepin production and bioconversion during C. militaris fermentation. We observed that porcine liver extracts increased cordycepin production. In addition, air supply (2 h/d) significantly increased the cordycepin level in surface liquid-cultured C. militaris after 14 days. Moreover, blue light light-emitting diode irradiation (16 h/d) increased cordycepin production. These findings indicated that these conditions are suitable for increasing cordycepin production. We used these conditions to obtain water extract from the mycelia of surface liquid-cultured C. militaris (WECM) and evaluated the anti-oral cancer activity of this extract in vitro and in vivo. The results revealed that WECM inhibited the cell viability of SCC-4 oral cancer cells and arrested the cell cycle in the G2/M phase. Oxidative stress and mitochondrial dysfunction (mitochondrial fission) were observed in SCC-4 cells treated with WECM for 12 hours. Furthermore, WECM reduced tumor formation in 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis through the downregulation of proliferating cell nuclear antigen, vascular endothelial growth factor, and c-fos expression. The results indicated that porcine liver extracts irradiated with blue light light-emitting diode and supplied with air can be used as a suitable medium for the growth of mycelia and production of cordycepin, which can be used in the treatment of oral cancer.

Uncovering global metabolic response to cordycepin production in Cordyceps militaris through transcriptome and genome-scale network-driven analysis.

The cellular metabolic adaptations of Cordyceps militaris have been progressively studied. In particular, the cordycepin pathway is of interest in medicinal applications. Even though the metabolic pathways for cordycepin production are known to be related to different carbon sources, the regulatory mechanisms at a systems level are poorly characterized. To explore the regulatory mechanisms, this study therefore aimed to investigate the global metabolic response to cordycepin production in C. militaris through transcriptome analysis and genome-scale network-driven analysis. Here, transcriptome analysis of 16,805 expressed genes in C. militaris strain TBRC6039 grown on different carbon sources was performed. Of these genes, 2,883 were significantly differentially expressed genes, uncovering sucrose- and glucose-mediated changes in the transcriptional regulation of central carbon metabolism in C. militaris, which was shown using the CmSNF1 mechanism as an example. After applying genome-scale metabolic network-driven analysis, reporter metabolites and key metabolic subnetworks involving adenosine, cordycepin and methionine were proposed through the up-regulation of cordycepin biosynthetic genes. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific culture conditions during cordycepin overproduction.

The effect of Zn on the Zn accumulation and biosynthesis of amino acids in mycelia of Cordyceps sinensis.

Zinc is an essential trace element in the human body and it participates in various pathways of metabolism. Cordyceps sinensis is a wellknown traditional Chinese medicine that contains cordycepin, cordycepic polysaccharides, proteins, vitamins, trace elements, and many other biological active materials. In this study, we cultured C. sinensis in liquid medium containing Zn ions and then analyzed the biomass, the ratios of total Zn accumulation, and the organic Zn content in the mycelia. The results showed that when the media contain 150 mg/L Zn, the biomass of mycelia of C. sinensis reached 10.7 g/L and the Zn content present in the mycelia reached 4875.1 microg/g. The percentage of Zn accumulated in the mycelia was 34.05% of the total Zn in the media, of which the organic Zn accounted for 76.33%. The results also revealed that the content of total amino acid (TAA) and essential amino acid (EAA) in the mycelia were increased substantially TAA and EAA in the Zn-treated mycelia were 11.1% and 15.2% higher, respectively, than that in the mycelia without Zn treatment. These results will be useful for elucidation of the physiological mechanism of Zn effects on the biological metabolism in the mycelia of C. sinensis.

Effects of cultural medium on the formation and antitumor activity of polysaccharides by Cordyceps gunnii.

The effects of culture medium composition (i.e., carbon and nitrogen sources) on the growth of mycelia, molecular weight distribution and antitumor activity of intracellular polysaccharides (IPS) from Cordyceps gunnii were investigated. Sucrose and peptone were proved to be the best carbon and nitrogen sources for mycelia growth and remarkably improved IPS production. When the sucrose concentration was 2.0%, the mycelium yield reached up to 15.94±1.26 g/L, but with lower IPS yield; whereas the sucrose concentration was 4.5%, IPS yield reached to a maximum of 138.78±3.89 mg/100 mL. The effects of different carbon/nitrogen (C/N) ratios with equal amounts of carbon source matter on the mycelia and IPS formation were optimized. It found that the yield of mycelia and IPS were both reached to the highest at a C/N ratio of 10:3. In addition, the IPS had the highest macro molecular polysaccharide content and antitumor activity when sucrose concentration was 3.5% and the C/N ratio was 10:1.5. Thus, there was a positive correlation between molecular weight distribution and antitumor activity of IPS by C. gunnii.

Medium optimization for polysaccharide production of Cordyceps sinensis.

As a potential anticarcinogenic agent, polysaccharides from Cordyceps sinensis have been demonstrated to possess strong antioxidation activity. The aim of the present research was to study the optimal medium to produce polysaccharides of C. sinensis by using response surface methodology (RSM). The composition of optimized medium for polysaccharide production calculated from the regression model of RSM was 6.17% sucrose, 0.53% corn steep powder, 0.5% (NH4)2HPO4, and 0.15% KH2PO4 at pH 4.44, with a predicted maximum polysaccharide production of 3.17 g/L. When applying this optimal medium, the maximum polysaccharide production was 3.05 and 3.21 g/L in a shake flask and a 5-L jar fermentor, respectively. When the pH was controlled at a higher level such as pH 5.0, both cell growth and polysaccharide production were inhibited. A low pH of 2.85 was required for maximum production of polysaccharides.