Effect of dietary supplementation with discardednutrient medium of Cordyceps militaris on layingperformance and diversity of intestinal flora in laying hens

The aim of the experiment was to explore the feasibility of discarded nutrient medium of Cordyceps militaris as feed in the production of laying hens. 100 g/kg, 200 g/kg, 300 g/kg of discarded nutrient medium of Cordyceps militaris were added to the basal diet of laying hens. The results showed that the optimal addition of discarded nutrient medium of Cordyceps militaris in the diet of laying hens was 10%. According to the results of measuring the conventional indicators of eggs, the weight of eggs produced by laying hens fed with discarded nutrient medium of Cordyceps militaris was higher than that of laying hens fed with ordinary laying hens. The content of interleukin-1(IL-1) in the experimental group was significantly higher than that in the control group, and the concentration of IL-1 increased by 141.5 pg/mL, which indicated that the application of discarded nutrient medium of Cordyceps militaris effectively improved the immunity of laying hens. The high-throughput analysis of the intestinal contents of the two groups of laying hens showed that the microbial population abundance of the intestinal tract of the experimental group was greater than that of control group, and the application of discarded nutrient medium of Cordyceps militaris increased the diversity of bacteria in the intestinal tract of laying hens. In addition, the sensitivity of some pathogenic bacteria in the intestinal tract of chickens to drugs was also increased, thereby reducing the use of antibiotics. The secondary utilization of discarded nutrient medium of Cordyceps militaris has great development and utilization prospects, which provided a scientific reference and basic theoretical basis for the development of discarded nutrient medium of Cordyceps militaris as feed in the production of laying hens.

Pharmacodynamic evaluation of Cordyceps sinensis (Berk.) Sacc. for ischemic stroke in rats and potential mechanism through network pharmacology and molecular docking

Objective:To explore the therapeutic potential of Cordyceps sinensis(Berk.)Sacc.(C.sinensis,Dong Chong Xia Cao)in an ischemic stroke(IS)model and predict its possible mechanism through network pharmacology.Methods:Thirty-three SpragueeDawley rats were randomly divided into the Sham,model,and C.sinensis groups.After 5 days of pre-treatment,the model group and the C.sinensis group were sub-jected to middle cerebral artery occlusion(MCAO)modeling.Effect of C.sinensis on MCAO rats was evaluated by comparing cerebral infarct size,neurological function,cerebral water content,pathological changes,and certain biochemical indicators.Intersection targets between C.sinensis and IS was screened using network pharmacology analysis.Relationship among core components,targets and pathways of C.sinensis in treating IS was constructed through network pharmacology analysis and further verified by molecular docking.Finally,the DAVID v8.8 database was used for performing GO analysis and KEGG pathway enrichment analysis by importing the intersection targets.Results:Compared with the model group,C.sinensis significantly reduced the volume of cerebral infarction(P=0.026),the cerebral water content(P¼.0013),the mNSS score(P<0.001),and the levels of IL-17(P=0.031),TNF-α(P=0.016),MDA(P=0.014),and glutamate(P=0.014)in serum,while upregulating the level of SOD in serum and improving the pathological morphology in MCAO rat ischemic brains.The results of network pharmacology analysis showed that core targets(such as CASP3,PTGS2,and PPARG)and the main enrichment pathways(IL-17,AGE-RAGE,and TNF signaling pathways)were regulated by 30 chemical components of C.sinensis,which effectively treated IS in MCAO rats.Conclusion:The results of this study showed that C.sinensis effectively interfered with MCAO rats,and the mechanism may be related to the regulation of blood lipids and to anti-apoptosis and anti-inflammatory effects.

Three previously undescribed metabolites from Cordyceps cicadae JXCH-1,an entomopathogenic fungus

Three previously undescribed compounds,cordycicadione(1),cordycicadin F(2),and 7-hydroxybassiatin(3),were isolated from the cultures of Cordyceps cicadae JXCH1,an entomopathogenic fungus.Their structures and relative configurations were elucidated primarily by NMR spectroscopic analysis.The absolute configurations of 1 and 2 were determined by ECD calculations.Single-crystal X-ray diffraction method was adopted to determine the absolute configuration of 3.Compound 2 is a polycyclic polyketide with an unusual enol ether moiety and a spiro ring.The compounds obtained in this study were subjected to screening their inhibition against the proliferation of the human lung cancer cell line A549 and the production of nitric oxide in murine macrophages RAW264.7.

Mechanism of Cordyceps sinensis in atherosclerosis treatment based on network pharmacology and molecular docking analysis

Background:The present study intented to delve into the molecular mechanism of Cordyceps sinensis(C.sinensis)in treating atherosclerosis by combining network pharmacology and molecular docking analysis.Methods:We searched the databases including Traditional Chinese Medicines Systems Pharmacology Database and Analysis Platform,PubChem,and PharmMapper to screen out the active chemical ingredients of C.sinensis and the corresponding targets.The String database was used for the matching normalization of results,and the software Cytoscape 3.7.2 was adopted to establish the C.sinensis-active components-targets of action-disease network.The databases of Online Mendelian Inheritance in Man database,GeneCards,Therapeutic Target Database,and DisGNET were searched to yield the major targets of atherosclerosis(AS),which were matched with the active component targets of C.sinensis to identify the potential therapeutic targets.The String database was utilized to set up the protein-protein interaction network,and Cytoscape software was applied for topological analysis,which was followed by the Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes signaling pathway analysis based on the DAVID database.Finally,the core components of C.sinensis and the targets of action were confirmed via molecular docking on AutoDock Vina and PyMOL.Results:In total,7 bioactive ingredients of C.sinensis were identified from Traditional Chinese Medicines Systems Pharmacology Database and Analysis Platform database and 319 predicted targets were obtained,231 of which were associated with AS.The core targets involved in AS treatment with C.sinensis included MAPK1,SRC,PIK3R1,AKT1,and HSP90AA1.The enrichment analysis unveiled the primary pathways involved in these processes,such as pathways in cancer and lipid and atherosclerosis.Moreover,through molecular docking,it was found that the active ingredients of C.sinensis presented with strong binding capacities with their corresponding targets,and the strongest binding capacity was observed between peroxyergosterol and SRC.Conclusions:The present study revealed at the molecular level that C.sinensis played its role in AS treatment through multiple drug active components,targets of action and pathways,which would point out the direction and provide theoretic basis for future research.

The Nucleosides Contents and Their Variation in Natural Cordyceps sinensis and Cultured Cordyceps Mycelia

Aim: To compare the contents of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia, and to study the effect of humidity and heat on the content of nucleosides. Methods: The contents of nucleosides were determined by using high performance capillary electrophoresis (HPCE). Beckman P/ACE System 5010 apparatus equipped with a UV detector and a Beckman untreated fused-silica capillary (57 cm 75 mm, 50 cm effective length) was used. Before sample injection, the capillary was rinsed with 1 molL-1 sodium hydroxide solution and running buffer for 5 min, respectively. A voltage of 20 kV was applied for the separation. Pressure injection was 586 kPa for 6 seconds, and the wavelength of detector was 254 nm. The running time was 20 min at 20 oC. The effect of humidity and heat on the contents of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia was observed for 1, 3, 5 and 10 days at temperature 40 oC, and relative humidity 75%. Results: The content of nucleosides from natural Cordyceps sinensis was higher than that from cultured Cordyceps mycelia. But the contents of nucleosides from freshly collected natural Cordyceps sinensis were very low, even below the limit of quantitation. The contents of nucleosides from natural Cordyceps sinensis were significantly increased by humidity and heat, but this phenomenon was not observed in cultured Cordyceps mycelia. Conclusion: There are differences between the nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia. The nucleosides in natural Cordyceps sinensis may be derived from the degradation of nucleic acids. This implies that adenosine being used for the quality control of natural Cordyceps sinensis may have to be reconsidered.

Polysaccharides Purified from Wild Cordyceps Activate FGF2/FGFR1c Signaling

Land animals as well as all organisms in ocean synthesize sulfated polysaccharides. Fungi split from animals about 1.5 billion years ago. As fungi make the evolutionary journey from ocean to land, the biggest changes in their living environment may be a sharp decrease in salt concentration. It is established that sulfated polysaccharides interact with hundreds of signaling molecules and facilitate many signaling transduction pathways, including fibroblast growth factor （FGF） and FGF receptor signaling pathway. The disappearance of sulfated polysaccharides in fimgi and plants on land might indicate that polysaccharides without sulfation might be sufficient in facilitating protein ligand/receptor interactions in low salinity land. Recently, it was reported that plants on land start to synthesize sulfated polysaccharides in high salt environment, suggesting that fungi might be able to do the same when ex- posed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chi- nese Medicine. One of the important pharmaceutical active ingredients in Cordyceps is polysaccharides. Therefore, we hypothesize that the salty environment inside caterpillar body might allow the fungi to synthesize sulfated polysaccharides. To test the hypothesis, we isolated polysaccharides from both lava and sporophore of wild Cordyceps and also from Cordyceps militaris cultured without or with added salts. We then measured the polysaccharide activity using a FGF2/FGFRlc signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccha- rides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

Investigation of Biological Characteristics of Parasitic Fusarium Fungi from Tibetan Cordyceps sinensis

[Objective] This study aimed to investigate the biological characteristics of parasitic Fusarium fungi from tibetan Cordyceps sinensis. [Method] A Fusarium strain was isolated from fresh tibetan Cordyceps sinensis as the experimental material, to observe the morphologies of Fusarium mycelium and spores. [Result] The isolated Fusarium fungus strain grew lushly on rice medium, with white and villiform myceliurn, and the villous stroma appeared in colors from beige to pink. After cultivated at （18 ± 4） ℃ for 14 d, the Fusarium strain produced milky white protrusions containing pale yellow chlamydospores, which can be divided into microconidiums and macroconidiums based on the different sizes. According to Ainsworth＇s classification system, the isolated strain belongs to Fusarium, Tuberculariales, Hyphomycetes, adelomycete subphylum. Further observation identified three types of macroconidium, and two of them showed distinct morphology, which was not described in Ainsworth＇s classification system. [Conclusion] The structure of the strain isolated from tibetan Cordyceps sinensis is significantly different from that of Fusariurn fungi recorded in relevant literatures, which requires further investigation.

The Effect of the Hot Water Extracts of the <i>Paecilomyces hepiali </i>and <i>Cordyceps militaris</i>Mycelia on the Growth of Gastrointestinal Bacteria

The gastrointestinal health is very important aspect concerning human health. It refers to nutrient and vitamin absorption, food digestion and various infectious diseases. The Paecilomyces hepiali and Cordyceps militaris are highly enriched with cordycepin and ergosterol which are considered as anti bacterial substances. Present study finds out comparative effect of hot water extract of particular fungal material on growth of six species of gastrointestinal bacteria that belong to both aerobic and anaerobic and, consist with harmful and commensal categories. The appropriate concentration level of hot water extract of both strains was identified. The individual specific bacterial growing media were prepared and calculated;amounts of bacteria cultures were inoculated by using micro pipettes. The optical density and number of bacterial colonies were measured after 24 hours. The pure mycelial extract of P. hepiali with 2 × 10-3 g/ml of concentration has significant effect on depleting the growth of E. coli, E. faecalis, S. aureus, L. gasseri and B. ovatus bacteria. B. longum has no significant effect by particular extract. Same type of extract of C. militaris has significantly reduced the growth of every bacteria used in this study. Hot water extract of C. militaris cultivated on soy bean has significant growth retardation toward E. coli, E. faecalis, S. aureus and L. gasseri. It has stimulated the growth of B. ovatus and B. longum which are considered as beneficial bacteria for human gut. This study shows that extracts of both mycelia include antimicrobial substances like cordycepin and ergosterol which can be used as food supplements to enhance human gut health.

Cultivated Cordyceps: A Tale of Two Treasured Mushrooms

Ophiocordyceps sinensis and Cordyceps militaris both contain many bioactive compounds that confer potential therapeutic benefits. This review discusses the possible use of cultivated C. militaris as an effective substitute for native O. sinensis in the face of ever-increasing prices of O. sinensis because of its short supply. On the one hand, cultivated C. militaris contains higher levels of cordycepin when compared with that of wild-type O. sinensis and cultivation of C. militaris has been shown to be capable of reducing the risk of heavy metal contamination. On the other hand, there is a paucity of robust in vivo studies and randomized controlled tests comparing the pharmacology and use of C. militaris and O. sinensis. For extraction of cordycepin as western-style tablets, the use of cultivated C. militaris rather than O. sinensis represents the most appropriate future approach. For many other purposes, comparative pharmacology and clinical trials are in urgent needs.

Optimization of Rice Medium for Cultivation of Cordyceps militaris in the Alpine Region

In this study,six Cordyceps militaris strains introduced to Tibet were used as experimental materials to screen the dominant C.militaris strain by comparing mycelial growth rate,fruiting body yield and growth status.The composition of rice medium for C.militaris cultivation was optimized by orthogonal experiment.According to the results,although C.militaris strain ＂TAAAS1＂ did not exhibit the highest mycelial growth rate,it had uniform fruiting body formation and higher fruiting body yield than other strains,which suggested that C.militaris strain ＂TAAAS1＂ was most suitable for cultivation and production of C.militaris in the alpine region.The optimal rice medium for C.militaris cultivation consisted of 4 g/L pupa powder,20 g/L glucose,1.5 g/L KH_2PO_4 and 2 g/L MgSO_4.This study provided the theoretical basis for cultivation and production of C.militaris in the alpine region.

Effects of Fungi Scratching on the Growth of North Cordyceps

[Objective] North Cordyceps is as valuable as the wild Cordyceps nensis on medicinal value, nutrients, and trace element contents, which attracts lots of researchers to explore the cultivation method. In order to master the best way to increase output, an experiment was conducted [Methods] To conclude the result, 100 boxes were chosen, of which 50 boxes were treated with fungiscratching in the same condition, based on comparisons of color changing and fruit body. [Results] The stroma have consistent length in good order ,but there is 1 box of scratching happened infections after operating. [Conclusion] Scratching can promote mycelium turning color better, shorter the cycle of fruiting, and prevent the phenomenon of ＂aside grass＂; and ＂no grass＂; effectively,al of this can make the North Cordyceps grow more dense and neatly.

Extraction, Purification and Anti-tumor Activity of Polysaccharide from Mycelium of Mutant Cordyceps Militaris

A water-soluble polysaccharide（named MCMP） was isolated from the mycelium with high yield mutation Cordyceps militaris by hot-water extraction, deproteinization by sevage, alcohol precipitation, anion-exchange and gel filtration chromatography CL-6B. The polysaccharide contained mannose, rhamnose, galactose and glucose in a molar ratio of 59.36：1：8.31：39.50, of which the average molecular weight is 8100. In our research, Hep-G2 cells, Hela cells and mesangial cells were chosen to determine the anti-tumor activity of the polysaccharide. The results of MTT assay show that polysaccharides of the mutant strain presented inhibitory activity on the cells proliferation after 48 h incubation.

Purification and Characterization of an Antibacterial Protein from the Cultured Mycelia of Cordyceps sinensis

An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein （CSAP）. CSAP was single-chained, with an apparent molecular mass of 35 × 10^3 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inbibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Further more, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.

Simultaneous determination of adenine, uridine and adenosine in cordyceps sinensis and its substitutes by LC/ESI-MS

A simple, sensitive and reproducible high performance liquid chromatography-mass spectrometry coupled with electrospray ionization method for simultaneous separation and determination of adenine, adenosine and （uridine） was developed. The analytical column is a 2.0 mm×150 mm Shimadzu VP-ODS column and volume fraction of the mobile phase is （86.5%）water, 12.0%methanol and 1.5%formic acid. 2-chloroadenosine was used as internal standard. Selective ion monitoring mode and selective ion monitoring ions at ratio of mass to electric charge of 136 for adenine, 268 for adenosine and 267 for uridine were chosen for quantitative analysis of the three active components. The results show that the regression equations and linear range are Y=0.062X+（0.005） and 2.0140.0 (μg·mL-1)for adenine, Y=0.049X+0.004 and 4.0115.0 μg·mL-1 for uridine, （Y=0.154X）+0.014 and （1.0125.0） μg·mL-1 for adenosine. The limits of detection are 0.6 μg·mL-1 for adenine, 1.0 μg·mL-1for uridine and (0.2 μg·mL-1) for adenosine. The recoveries of the three constituents are from 96.6% to 103.2%.

Isolation and Purification of Polysaccharides from Cordyceps minlitaris and Its Inhibition on the Proliferation of Rat Glomerular Mesangial Cells

The crude polysaccharide was obtained by means of the decolorization of porphyrized Cordyceps minlitaris stroma with organic solvent, extraction with hot water, precipitation in 80% ethanol, and protein removal with the Sevag method. After purification with Sephadex G-75, two of its components, CMP-1 and CMP-2, were obtained. Through the assay of gel chromatography and polarimetry, CMP-1 was identified as pure polysaccharide. The results demonstrated that CMP-1 had favorable oxidation resistance activity, which could scavenge not only oxygen-free radicals in the self-oxidation system of pyrogallic acid, but also the hydroxide-free radicals in the Fenton system. The study focused on the effects of low, medium, and high dosages of CMP-1 in rat blood serum on the proliferation of glomerular mesangial cells in vitro. Through MTT Colorimetric analysis, the activities were compared among the blank control group and the Niaoduqing positive control group CMP-1 and CMP-2. The results shows that CMP-1 was able to inhibit the proliferation of rat glomerular mesangial cells effectively. Therefore, CMP-1, one component of polysaccharides of Cordyceps minlitaris, was certainly a potential remedy for hyperplastic glomerular nephritis, whose antioxidant activity could slow down the process of chronic renal failure（CRF） to some extent.

Isolation of Cordyceps ophioglossoides L2 from Fruit Body and Optimization of Fermentation Conditions for Its Mycelial Growth

A strain isolated from the fruiting body of a fungus parasitized on Elaphomyces was identified as Cordyceps ophioglossoides based on the morphological characteristics and the analysis of ITS-5.8s rDNA sequence. The optimal medium, composition （g·L^-1）, containing sucrose 66.0, yeast powder 10.0, silkworm chrysalises digest 30.0, MgSO4· 7H2O 0.4, and KH2PO4 0.4, Was found using fractional factorial design ancl a central composite design, and the optimization of cultural conditions obtained a result of seed age 6 days, inoculum size 6% （by volume）, initial pH 5.6, temperature 24℃, shaking speed 160 ·＇min^-1 by one-factor-at-a-time method. The maximum biomass reached about 20.2 g·L^-1 after 90 hours culture under the optimal conditions. Elementary nharmaeclogical actlwtties showed that mycelia of C. ophioglossoides L2 from submerged culture promoted Uterus growth in estrogen- depleted mice. In the 15-litre scale-up fermentation, the mycelial biomass was around 19.1 g·L^-1, indicating a promising prospect for this biotechnoloagy and the potency to develoo its medical value.

Randomized Double-blind Placebo-controlled Clinical Trial andAssessment of Fermentation Product of Cordyceps Sinensis (Cs-4) inEnhancing Aerobic Capacity and Respiratory Function ofThe Healthy Elderly Volunteers

Objective:Cordyceps sinensis (CS) is a popular natural Chinese herbal medicine for invigoration, health preservation and reducing fatigue. Its natural substance has been prepared as a fermentation product of a specific strain of Cordyceps sinensis (Cs-4). Our objective was to assess the effect of Cs-4 on the exercise capacity of the healthy elderly people in a randomized, double-blind, placebo-controlled trial. Methods:Thirty-seven healthy, elderly Chinese subjects were randomly assigned to receive either Cs-4 (3 g/ day) or identical placebo capsules. Their exercise performance was tested before and after 6 weeks of treatment with a symptom-limited, incremental work rate protocol on a cycle ergometer. Maximum oxygen uptake (VO2max) was measured using a metabolic chart. Anaerobic thresholds (VO26) were identified by two observers using plots of both VCO2 vs VO2 and VE/VO2 vs time. Results: After taking Cs-4 for 6 weeks, VO2max (1 88±0.13 to 2.00±0 14 L/min; P=0.050) and VO2(1.15±0.07 to 1.30±0.09 L/min; P = 0.012) were significantly increased, whereas after placebo application they were unchanged. Conclusion: These findings support the belief held in China that Cs-4 could improve oxygen uptake or aerobic capacity and ventilation function and resistance to fatigue of elderly people in exercise.

Determination of Cordycepin in Cordyceps kyushuensis by Capillary Electrophoresis and its Antitumour Activity

A simple, rapid and low-cost method of determination for cordycepin in Cordyceps kyushuensis by capillary zone electrophoresis (CZE) was developed. Based on the finding that there is a high concentration of cordycepin in both natural and cultured Cordyceps kyushuensis, the in vitro antitumor activity of cordycepin and the water extracts of Cordyceps kyushuensis has been investigated. This is the first report about the antitumor effect of Cordyceps kyushuensis.

Studies on Structure and Properties of Water Soluble Polysaccharide from Fruiting Body of Cordyceps Militarvs(L.) Link

A water soluble crude polysaccharide has been isolated from the fruiting body of cultivated Cordyceps militarvs(L.) Link. The GC analysis indicated that it contained the monosaccharides of Gal, Man, Glc and GlcA. Their molar ratio is 1 00∶1 58∶7 89∶0 19. The crude polysaccharide was fractionated with ethanol to get three fractions, which were CM Ⅰ, CM Ⅱ and CM Ⅲ. CM Ⅱ was further purified to get one homogeneous fraction named CMB. The molecular weight was estimated to be 60000 by means of gel filtration. CMB is a greyish white powder. The GC analysis indicated that it was composed of Gal, Man, Glc and the molar ratio is 1 00∶1 38∶5 10. Partial hydrolysis with an acid, periodate oxidation, Smith degradation, IR, GC and methylation analyses, GC/MS analyses were used for the structure analyses of CMB. The results reveal that CMB is of a lower branched structure. The main chain is composed of (1→6) linked Man, (1→6) linked Glc, (1→4) linked Glc and (1→4) linked Gal. Some of (1→4) linked Glc and all (1→4) linked Gal residues are substituted at 3 O and 6 O respectively. (1→4) Linked Glc residues without branch probably exist with other monosaccharides alternately. On an average, there is one branch every ten hexose residues. The side chain is composed of major (1→4) linked Glc and minor (1→6) linked Glc. The nonreduced end is composed of Gal and Glc. The main glycosidic link of CMB is α configuration.

Optimization for Production of Intracellular Polysaccharide from Cordyceps ophioglossoides L2 in Submerged Culture and Its Antioxidant Activities in vitro

Cordyceps ophioglossoides is a valuable traditional medicinal material.We have found that intracellular polysaccharide(IPS) is the major biologically active ingredient in Cordyceps ophioglossoides.This study is the first time to optimize the yield of IPS from Cordyceps ophioglossoides.The optimal medium for IPS production consists of glucose 54.50 g·L·1,yeast powder 25.50 g·L·1,NaH2PO4 0.4 g·L·1 and K2HPO4 0.4 g·L·1.The suggested culture conditions are 24 ℃,initial pH 4.5 with a rotary speed of 120 r·min·1 for 168 h.The yield of IPS is 737.93 mg·L·1,which is 50% higher than the yield under the conditions prior to optimization.The anti-oxidative activities of IPS in Cordyceps ophioglossoides L2 are also characterized using various in vitro assay.The anti-oxidative activity may explain the reason why IPS from Cordyceps ophioglossoides can be used to fight against neurodegenerative dis-eases and menopausal symptoms.