Wild Cordyceps Research Articles

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 fungi 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 exposed in such environment. Interestingly, Cordyceps, a fungus habituating inside caterpillar body, is the most valued traditional Chinese 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/FGFR1c signaling-dependent BaF3 cell proliferation assay and found that polysaccharides isolated from wild Cordyceps activated FGF2/FGFR signaling, indicating that the polysaccharides synthesized by wild Cordyceps are indeed different from those by the cultured mycelium.

Effects of heat on the biological activity of wild Cordyceps sinensis

BackgroundCurrent methods of extending the storage time of wild Cordyceps sinensis adversely affect the nutritive and medicinal value of the product. Thus, this study was designed to investigate the effects of heat treatment, a relatively safe storage extension method, on the biological activity of wild C. sinensis. MethodsSamples were heated to 60, 80, or 100°C for 15, 30, or 60 minutes. SOD activity in wild C. sinensis before and after heating was assayed using a standard colorimetric assay. Deoxyribonuclease (DNase) activity was measured using the plasmid-nicking assay. Cordycepin content was analyzed using HPLC. Polysaccharide content was measured using the phenol-sulfuric method. The Student's t-test was used for comparison. ResultsAfter heating at 60, 80, 100°C for 15, 30, 60 minutes, respectively, no significant reduction in DNase activity or polysaccharide dissolution was noted (P > .05). Interestingly, heating at 80°C for 30 minutes led to a significant increase in the SOD activity of C. sinensis (P < .05). In addition, heating at 60°C for 60 minutes or at 80°C for 15 minutes significantly increased cordycepin dissolution (P < .05). Other heat treatments had no significant effects on SOD activity or cordycepin dissolution (P > .05). ConclusionsThese results suggested that heat treatment does not adversely affect SOD or DNase activity, polysaccharide content, or cordycepin dissolution. Thus, heat treatment might be a safe processing method to extend the storage time of wild C. sinensis without compromising biological activity.

Salinity-induced anti-angiogenesis activities and structural changes of the polysaccharides from cultured Cordyceps Militaris.

Cordyceps is a rare and exotic mushroom that grows out of the head of a mummified caterpillar. Many companies are cultivating Cordyceps to meet the increased demand for its medicinal applications. However, the structures and functions of polysaccharides, one of the pharmaceutical active ingredients in Cordyceps, are difficult to reproduce in vitro. We hypothesized that mimicking the salty environment inside caterpillar bodies might make the cultured fungus synthesize polysaccharides with similar structures and functions to that of wild Cordyceps. By adding either sodium sulfate or sodium chloride into growth media, we observed the salinity-induced anti-angiogenesis activities of the polysaccharides purified from the cultured C. Militaris. To correlate the activities with the polysaccharide structures, we performed the 13C-NMR analysis and observed profound structural changes including different proportions of α and β glycosidic bonds and appearances of uronic acid signals in the polysaccharides purified from the culture after the salts were added. By coupling the techniques of stable 34S-sulfate isotope labeling, aniline- and D5-aniline tagging, and stable isotope facilitated uronic acid-reduction with LC-MS analysis, our data revealed for the first time the existence of covalently linked sulfate and the presence of polygalacuronic acids in the polysaccharides purified from the salt added C. Militaris culture. Our data showed that culturing C. Militaris with added salts changed the biosynthetic scheme and resulted in novel polysaccharide structures and functions. These findings might be insightful in terms of how to make C. Militaris cultures to reach or to exceed the potency of wild Cordyceps in future.

Selenium Enrichment on Cordyceps militaris Link and Analysis on Its Main Active Components

To investigate the effects of selenium on the main active components of Cordyceps militaris fruit bodies, selenium-enriched cultivation of C. militaris and the main active components of the fruit bodies were studied. Superoxide dismutase (SOD) activity and contents of cordycepin, cordycepic acid, and organic selenium of fruit bodies were sodium selenite concentration dependent; contents of adenosine and cordycep polysaccharides were significantly enhanced by adding sodium selenite in the substrates, but not proportional to sodium selenite concentrations. In the cultivation of wheat substrate added with 18.0ppm sodium selenite, SOD activity and contents of cordycepin, cordycepic acid, adenosine, cordycep polysaccharides, and total amino acids were enhanced by 121/145%, 124/74%, 325/520%, 130/284%, 121/145%, and 157/554%, respectively, compared to NS (non-selenium-cultivated) fruit bodies and wild Cordyceps sinensis; organic selenium contents of fruit bodies reached 6.49mg/100g. So selenium-enriched cultivation may be a potential way to produce more valuable medicinal food as a substitute for wild C. sinensis.

Fatty acid composition of lipids in wild Cordyceps sinensis from major habitats in China

Wild Cordyceps sinensis (WCS) is considered to be one rare and valuable medicinal fungus for preventing and treating diverse chronic diseases, and its quality and price vary hugely with different producing areas. However, the determination of geographic origin has long been hindered by uncertainties of existing methods. Lately, it has been found that the fatty acid composition in lipids may be used to access geographic origin. Seventeen fatty acids were identified and their relative and absolute contents were measured by gas chromatography−mass spectrometry (GC−MS) in these samples with C16:0, C18:0, C18:1 and C18:2 as major constituents including linolenic acid (C18:3) (1.02−4.83%). Total unsaturated fatty acids (75.74−90.87%) are evidently enriched compared with the other common fungi. Based on the specific fatty acid composition in lipids, the influences of environmental temperature and humidity on lipid synthesis in WCS are discussed, and the fatty acid profile and the ratio of C18:1/C18:2 in polar lipids are suggested to be two potential indicators for determination of geographical origin.

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, 1H NMR and 13C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α- d-glucose residues (∼77%) and (1 → 6)-α- d-mannose residues (∼23%). Branching occurred at O-3 position of (1 → 6)-α- d-mannose residues of the backbone with (1 → 6)-α- d-mannose residues and (1 → 6)-α- d-glucose residues, and terminated with β- d-galactose residues.

Antioxidant activity of aqueous extract of a Tolypocladium sp . fungus isolated from wild Cordyceps sinensis

Tolypocladium sp. Ts-1 was isolated from the fruiting body of a wild Cordyceps sinensis, one of the best known traditional Chinese medicine and health foods. The antioxidant activities of hot-water extracts from cultured mycelia of Tolypocladium sp. were assessed in different in vitro systems. The extracts showed superoxide dismutase (SOD) activity of 35.6 U/mg protein and are effective in scavenging superoxide radical in a concentration dependent fashion with IC50 value of 1.3 mg/mL. As a reinforcement of the action, similar radical scavenging effects of the extracts were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. 1,1-Diphenyl-2-picrylhydracyl (DPPH) radical scavenging activities of the extracts reached more than 75.2% at the concentrations of 3-6 mg/mL. The extracts showed moderate reducing power and ferrous ion chelating activity. Moreover, the protective effects of the extracts against hydrogen peroxide (H2O2)-induced lesion to rat PC12 cell (pheochromocytoma cell line) was observed in a dose-dependent manner from 0.25 to 2.0 mg/mL. Our results suggest that the aqueous extract of Tolypocladium sp. mycelium has strong antioxidant activities and is a potential source of natural antioxidant products. This is the first report on antioxidant activity of Tolypocladium fungus isolated from wild C. sinensis, an endangered species. Key words: Aqueous extracts, antioxidant activity, free radicals, PC12 cells,Tolypocladium sp.

Mycelium cultivation, chemical composition and antitumour activity of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium. The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22-25 degrees C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1.1 day(-1), reaching a cell density of 23.0 g dw l(-1) in 7-9 days. Exopolysaccharides accumulated in the liquid culture to about 0.3 g l(-1) glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11.7-microg) and carbohydrate (654.6-microg) but much higher contents of polysaccharide (244.2 mg vs 129.5 mg), adenosine (1116.8-microg vs 264.6 microg) and cordycepin (65.7 microg vs 20.8 microg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour growth in mice. The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated by liquid fermentation. The mycelium biomass contained the major bioactive compounds of C. sinensis, and the mycelium extract had significant antitumour activity. The Cs-HK1 fungus may be a new and promising medicinal fungus and an effective and economical substitute of the wild C. sinensis for health care.

Differentiation of medicinal Cordyceps species by rDNA ITS sequence analysis.

Chongcao (the sexual stage of Cordyceps sinensis) is an important traditional Chinese fungal drug commonly used as a tonic and/or drug for hundreds of years. Recently, due to an increased demand for Cordyceps sinensis, the isolation of the fungus from the environment has been insufficient, and so other species of Cordyceps or artificially cultured Cordyceps sinensis mycelium as a conidial form (asexual stage) has been used to substitute for wild Cordyceps sinensis. There are more than ten Cordyceps species, and their related asexual species that also carry the name Chongcao on commercial markets. Since the value of each Chongcao may differ by more than 100-fold, the authentication of Chongcao is essential to secure their efficacy. However, the identification of the origin of a particular species, especially of its bio-products on the market, is difficult. Here, DNA sequence analysis of the rDNA ITS (internal transcribed spacer) was exploited to differentiate the medicinal species Cordyceps sinensis from several related Cordyceps species, such as, e. g., C. militaris, C. gunnii and their asexual stages. The data demonstrate that the rDNA ITS sequences of C. sinensis collected from different geographical regions are almost identical and are significantly different from the substitutes, so that they could be easily distinguished at the DNA level. Furthermore, based on the highly divergent ITS region of rDNA, a pair of species-specific primers were designed and used for the rapid identification of Cordyceps sinensis.