Cordyceps Pruinosa Research Articles

Spore Viability and Cell Wall Integrity of Cordyceps pruinosa Treated with an Electric Shock-Free, Atmospheric-Pressure Air Plasma Jet

Atmospheric-pressure A r plasma jets are known to be detrimental to Cordyceps pruinosa spores. However, it is not clear what kinds of reactive species are more effective with regard to fungal cell death. Herein, we study which reactive species plays pivotal roles in the death of fungal spores using an electric shock-free, atmospheric-pressure air plasma jet, simply called soft plasma jet. Plasma treatment significantly reduced the spore viability and damaged fungal DNA. As observed from the circular dichroism spectra, scanning electron microscope images, and flow cytometric measurements, cell wall integrity was decreased by reactive oxygen and nitrogen species (RONS) from the plasma itself and the plasma-activated water. Consequently, degradation of the spore cell wall allows RONS from the plasma to reach the intracellular components. Such plasma-induced intracellular RONS can attack spore DNA and other intracellular components, as confirmed by electrophoresis analysis and phosphorylated histone measurement. In addition, weakening of the spore cell wall allowed for the loss of intracellular components, which can lead to cell death. Plasma radicals were investigated by measuring the optical emission spectrum of the soft plasma jet, and intracellular reactive oxygen species were confirmed by measuring the fluorescence of 2′, 7′-dichlorodihydrofluorescein-diacetate ( H 2 D C F - D A )-stained spores. The soft plasma jet generated considerable amounts of H 2 O 2 and N O x but a very small number of O H radicals as compared to the atmospheric-pressure A r plasma jet; this indicates that plasma-induced long-lived reactive species ( H 2 O 2 and N O x ) play an important role in the weakening of spore cell walls and cell death.

Beauveria bassiana: a new N6-(2-hydroxyethyl)-adenosine–producing fungus

ABSTRACTN6-(2-Hydroxyethyl)-adenosine (HEA), which was the first calcium antagonist derived from biological sources, has been intensively investigated because of its ability to inhibit tumour cell proliferation, restrain inflammation, protect kidneys and function as a sedative and insecticide. Up to now, the production of HEA has been detected only in a few species such as Cordyceps pruinosa, C. militaris and Isaria tenuipes. Here, we found a new HEA-producing fungus, which was identified as Beauveria bassiana based on morphological and phylogenetic characteristics. The HEA production was verified by reversed-phase high-performance liquid chromatography/mass spectrometry in this fungus. Moreover, HEA was also detected in the mycelia of two other B. bassiana strains from different origins, but not in the culture medium of all tested B. bassiana strains. The maximum production of HEA (0.8483 ± 0.0439 mg/g mycelia dry weight) was achieved on day 7. The fungal biomass and kinetics of HEA production exhibited similar trends over the duration of the culture period. This is the first report describing HEA production in B. bassiana, which suggests that this fungal strain may have new applications as a source of HEA.

Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds.

Cordyceps sensu lato is a genus of arthropod-pathogenic fungi, which have been used traditionally as medicinal in Asia. Within the genus, Ophiocordyceps sinensis is the most coveted and expensive species in China. Nevertheless, harvesting wild specimens has become a challenge given that natural populations of the fungus are decreasing and because large-scale culture of it has not yet been achieved. The worldwide demand for products derived from cultivable fungal species with medicinal properties has increased recently. In this study, we propose a new species, Cordyceps nidus, which parasitizes underground nests of trapdoor spiders. This species is phylogenetically related to Cordyceps militaris, Cordyceps pruinosa, and a sibling species of Cordyceps caloceroides. It is found in tropical rainforests from Bolivia, Brazil, Colombia and Ecuador. We also investigated the medicinal potential of this fungus based on its biochemical properties when grown on four different culture media. The metabolic profile particularly that of nucleosides, in polar and non-polar extracts was determined by UPLC, and then correlated to their antimicrobial activity and total phenolic content. The metabolome showed a high and significant dependency on the substrate used for fungal growth. The mass intensities of nucleosides and derivative compounds were higher in natural culture media in comparison to artificial culture media. Among these compounds, cordycepin was the predominant, showing the potential use of this species as an alternative to O. sinensis. Furthermore, methanol fractions showed antimicrobial activity against gram-positive bacteria, and less than 3.00 mg of gallic acid equivalents per g of dried extract were obtained when assessing its total phenolic content by modified Folin-Ciocalteu method. The presence of polyphenols opens the possibility of further exploring the antioxidant capacity and the conditions that may enhance this characteristic. The metabolic composition and biochemical activity indicate potential use of C. nidus in pharmaceutical applications.

한국에서 개발된 곤충유래 약용버섯인 누에동충하초의 생산기술개발 및 약리학적 특성

Cordyceps is a traditional Chinese medicinal herb well-known in China, Korea and Japan since B.C. 2,000. The original entomopathogenic fungus, Cordyceps sinensis belonging to the genus Cordyceps could not be found inside Korean peninsula due to the absence of the host insect for the corresponding entomogenous fungus. The development of artificial production methods of Korean type Cordyceps using the silkworm Bombyx mori as in vivo culture medium for the the entomopathogenic fungus Paecilomyces tenuipes is the first, and wonderful occasion in the research history of insect industry of this global world. The aim of this article is to review the historical research background, mass-production methods, and pharmacological effects of the silkworm-dongchunghacho (Paecilomyces tenuipes) which is a newly developed Korean medicinal insect-borne mushroom, and another non-insect-borne medicinal mushroom (Cordyceps militaris and Cordyceps pruinosa). Their biological actions include anti-tumor, immunostimulating, anti-fatigue, anti-stress, anti-oxidant, anti-aging, anti-diabetic, anti-inflammatory, anti-thrombosis, hypolipidaemic and insecticidal effects. The bioactive principles are protein-bound polysaccharides (hexose, hexosamin), cordycepin, D-manitol, acidic polysaccharide etc. Protein-bound polysaccharides and n-butanol fractions were demonstrated to show a significant anti-tumor activities but did not show a cytotoxicities. D-mannitol exhibited a significant prolongation of the life span in tumor bearing mice. Ergosterol did not show an efficient anti-tumor activity, but showed a significant phagocytosis enhancing activity. Anti-tumor activity of silkworm-dongchunghacho might be attributed to immuno-stimulating activities rather than cytotoxic effects [164]. Also this review comprises the breeding of Dongchunghacho varieties, optimization of culture conditions, improvement of learning and memory by Dongchunghacho, application of them as foods and chemical constituents.

Effects of reactive oxygen species on the biological, structural, and optical properties of Cordyceps pruinosa spores

Effects of reactive oxygen species on the biological and optical properties of Cordyceps pruinosa spores were studied. The decline in spore viability, antioxidative capacity, and DNA content were due to structural alteration of the cell wall.

Cordycepin and N6-(2-hydroxyethyl)-adenosine from Cordyceps pruinosa and their interaction with human serum albumin.

Cordyceps pruinosa (CP) is often used as Traditional Chinese Medicine, but the substance basis of its medicinal properties is unclear. In this study, two compounds were isolated from CP cultures by column chromatography, and identified as cordycepin and N6-(2-hydroxyethyl)-adenosine (HEA) by Nuclear Magnetic Resonance. In order to understand the efficacy of these two substances as potential therapeutic agents, it is necessary to explore their binding with proteins. The molecular mechanisms of interaction between cordycepin, HEA and human serum albumin (HSA) were studied using UV and fluorescence spectroscopy. The bingding constants between HSA and cordycepin were 4.227, 3.573 and 3.076 × 103·at 17, 27 and 37°C respectively, and that of HSA and HEA were 27.102, 19.409 and 13.002 × 103·at the three tempretures respectively. Both cordycepin and HEA can quench the intrinsic fluorescence of HSA via static quenching, and they can bind with HSA to form complexes with a single binding site. The interaction forces between cordycepin and HSA were determined as electrostatic and hydrophobic, and those of HEA and HSA were hydrogen bonding and van der Waals forces. Using Foster's equation, the distance between fluorophores of cordycepin and HSA, and HEA and HSA are estimated to be 5.31 nm and 4.98 nm, respectively. In this study, cordycepin was isolated for the first time from CP, and will provide a new source of cordycepin and expand the use of this taxon. The interaction mechanisms between cordycepin and HSA was studied for the first time, which will provide a useful guide for the clinical application of cordycepin. The pharmacological importance of this study is to understand the interaction of HSA with cordycepin and HEA, which will be essential for the future designing of drugs based on the two compounds.

NMR and GC-MS based metabolic profiling and free-radical scavenging activities of Cordyceps pruinosa mycelia cultivated under different media and light conditions.

Variation of metabolic profiles in Cordyceps pruinosa mycelia cultivated under various media and light conditions was investigated using 1H nuclear magnetic resonance (NMR) analysis and gas chromatography mass spectrometry (GC-MS) with multivariate statistical analysis. A total of 71 metabolites were identified (5 alcohols, 21 amino acids, 15 organic acids, 4 purines, 3 pyrimidines, 7 sugars, 11 fatty acids, and 5 other metabolites) by NMR and GC-MS analysis. The mycelia grown in nitrogen media and under dark conditions showed the lowest growth and ergosterol levels, essential to a functional fungal cell membrane; these mycelia, however, had the highest levels of putrescine, which is involved in abiotic stress tolerance. In contrast, mycelia cultivated in sabouraud dextrose agar with yeast extract (SDAY) media and under light conditions contained relatively higher levels of fatty acids, including valeric acid, stearic acid, lignoceric acid, myristic acid, oleic acid, palmitoleic acid, hepadecenoic acid, and linoleic acid. These mycelia also had the highest phenolic content and antioxidant activity, and did not exhibit growth retardation due to enhanced asexual development caused by higher levels of linoleic acid. Therefore, we suggested that a light-enriched environment with SDAY media was more optimal than dark condition for cultivation of C. pruinosa mycelia as biopharmaceutical or nutraceutical resources.

Cordyceps pruinosa produces cordycepin and N6-(2-hydroxyethyl)-adenosine in culture

Cordyceps species are entomophagous pathogens with medicinal properties, mostly linked to cordycepin and N6- (2-hydroxyethyl)-adenosine (HEA). An isolate of Cordyceps pruinosa (GZUCC 8552) was obtained from a fruiting body formed on the cocoon a Limacodidae insect collected in Guizhou Province, China. Morphological and molecular analysis (combined 5.8S ITS, RPB1 and 18S RNA) confirmed the species to be Cordyceps pruinosa. Metabolites of the isolate grown in liquid static and solid-state media were established by HPLC-MS. Cordycepin (5.311 mg/g) and HEA (0.558 mg/g) were produced by this strain. This is the first record of cordycepin from an isolate of Cordyceps pruinosa. As Cordyceps pruinosa is a good source of cordycepin and HEA, it could be used as an alternative to the over-collected Cordyceps sinensis.

Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway

Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa. Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used. CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants. These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.

Cordyceps pruinosa induces apoptosis in HeLa cells

Cordyceps is a parasitic fungus and has long been used as a Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and anticancer. In this study, especially, we focused on the mechanisms of cell death exhibited by Cordyceps pruinosa butanol fraction (CPBF) against human cervical adenocarcinoma cell line, HeLa. CPBF inhibited growth and induced apoptosis in HeLa cells in a dose‐dependent manner as determined by MTT assay, DNA fragmentation and phosphatidyl serine distribution. In western blot analysis, apoptosis in Hela cells was associated with the activation of caspase‐3, ‐9 and proteolytic cleavage of PARP, decrease of Bcl‐2/Bax protein ratio, and release of cytochrome C from mitochondria into cytosol. Apoptosis induced by CPBF could not be prevented by various antioxidants. These results show that apoptosis of CPBF toward HeLa cells is mediated by mitochondria‐dependent death‐signaling pathway regardless of reactive oxygen species. Thus, it indicated that CPBF may be a potential candidate in future research and development of bioactive ingredients as anticancer agents. Supported by grant from the BioGreeen 21 program, Korea

이축 압출 성형기를 이용한 붉은자루 동충하초의 압출 성형

The extrusion-cooking condition on Cordyceps pruinosa was designed using twin-screw extruder. Response surface methodology (RSM) was used to investigate extrusion-cooking using a central composition design with varying die temperature , feed moisture , feed rate (4-14 ka/h) and screw speed (120-280 rpm). System parameters (die pressure and specific mechanical energy (SME)) and extrudate parameters (density and water solubility index (WSI)) were statically analyzed using RSH. Die pressure was significantly affected by temperature, moisture contents and feed rate. SM was affected by screw speed and feed rate. When die temperature is and moisture content , the optimum pressure is shown. SME is about 20 Wh/kg, when feed rate is and screw speed . WSI was affected by temperature and moisture contents. Density was not affected by any factor. WSI increases by from about to about , as temperature is raised from . The WSI of Cordyceps pruinosa pulverized after extruding (PE) is about higher than that of raw material and higher than that of pulverized after drying (PD). The content of unsaturated fatty acid were not significantly different in PD and PE. Anti-oxidative activity of PE was 1.67-2.2 times higher than that of PD in Cordyceps pruinosa using 1- dipheny1-2-picrylhydrazyl method (DPPH).

한국에 자생하는 붉은자루동충하초(Cordyceps pruinosa)의 분포와 균사생장에 적합한 조건

붉은자루동충하초는 땅속의 죽은 풀쐐기 원형 번데기를 기주로 하여 1개 또는 <TEX>$3{\sim}4$</TEX>개의 곤봉형 자좌를 형성한다. 붉은색을 띠는 자좌는 머리와 자루의 경계가 명확하며 머리는 곤봉형이다. 크기는 <TEX>$1{\sim}3\;cm$</TEX>의 크기로 반묻힌형의 자낭각이 조밀하게 분포하며. 자낭각의 크기는 <TEX>$360{\sim}400{\times}180{\sim}200\;{\mu}m$</TEX>이다. 자낭은 <TEX>$150\;{\mu}m$</TEX>의 크기를 가지며, 둘레는 <TEX>$2.8{\sim}3.5\;{\mu}m$</TEX>이다. 중앙에 실모양의 끈으로 연결되어 있는 양끝에 자낭포자를 형성하며, 크기는 <TEX>$124{\sim}141\;{\mu}m$</TEX>의 크기를 가진다. 자낭포자는 2차 포자로 분열하지 않으며, 자낭포자의 각 세포들이 직접 발아하여 균사로 된다. 불완전세대는 Mariannaea속으로 분생자경의 4개<TEX>${\sim}$</TEX>5개의 파일라이드를 가졌고 크기가 <TEX>$15{\sim}24{\times}2{\sim}3\;{\mu}m$</TEX>이며 분생포자가 둥근 방추형이며 크기가 <TEX>$4{\sim}6{\times}1.8{\sim}2.4{\mu}m$</TEX>인 것으로 보아 Mariannaea elegans Samson으로 동정하였다. 균사의 생장은 YMA와 SDAY에 pH 7로 맞추고 <TEX>$25^{\circ}C$</TEX>로 배양하면 균사의 생장도 좋았고 균총의 밀도도 높았다. 탄소원은 Dextrin에서 가장 좋은 균사 생장과 균체량이 많았으며 다음으로 Lactose, Saccharose와 sucrose에서 균사 성장이 좋았으며 질소원은 peptone, yeast extract, trypeptone에서 균사 생장과 액체배지의 균사량도 많았다. C/N비는 1:1의 비율에서 균사의 생장과 밀도가 양호하였으며 적정량은 12.5 g/1에서 균사의 생장과 밀도가 양호하였다. 무기염류에서는 <TEX>$KH_2PO_4$</TEX>가 다른 무기염류에 비해 좋게 나타났다. 붉은자루동충하초의 대량 배양을 위하여 액체배지로는 YMA와 SDAY 배지에서 가장 많은 건조 균체량을 얻을 수 있었으며, 이 배지에 6개의 절편을 넣고 7일간 배양하면 많은 건조 균체량를 얻을 수 있었다. Cordyceps pruinosa grows upon dead pupae of Lepidoptera and produces one or <TEX>$3{\sim}4$</TEX> club-shaped stromata per host. The stromata have distinct club-shaped head and long stalk. The length of stromata varies from <TEX>$1{\sim}3\;cm$</TEX>. Apical head consists of densely crowded semi-immersed perithecia, which are <TEX>$360{\sim}400\;{\times}\;180{\sim}200\;{\mu}m$</TEX> in size. Asci are <TEX>$150\;{\mu}m$</TEX> in length and <TEX>$2.8{\sim}3\;{\mu}m$</TEX> in diameter. Ascospores, which are <TEX>$124{\sim}141\;{\mu}m$</TEX> in length, have thin thread-like structures in the middle with part-spores attached on both sides. Each ascospore does not separate into part-spores after dispersal, but each part-spore germinates and together develops a colony. The imperfect form produces phialides of <TEX>$15{\sim}24\;{\times}\;2{\sim}3\;{\mu}m$</TEX> size, with spherical or spindle shaped conidia of <TEX>$4{\sim}6\;{\times}\;1.8{\sim}2.4\;{\mu}m$</TEX> size, The anamorph was identified as Mariannaea elegans Samson. YMA and SDAY agar media with pH 7 was produced abundant mycelial growth with high density. Best mycelial growth was observed when dextrin was used as a carbon source. Lactose, saccharose and sucrose also produced high mycelial growth. Peptone, yeast extract and tryptone produced abundant mycelial growth, when used as nitrogen sources. Highest mycelial growth and density was observed when C/N ratio was 1 : 1 at the concentration of 12.5 g/l each. <TEX>$KH_2PO_4$</TEX> was the best mineral source for mycelial growth. Highest mycelial dry wt. was produced in YM and SDAY broths. Optimum inoculum for 100 ml of liquid broth was 6 mycelial discs. Similarly, optimum liquid culture period was 7 days.

Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa

Cordyceps pruinosa is an entomogenous fungus noteworthy for its various bioactivities. The influence of synthetic medium and cultural conditions on polysaccharides production was investigated in shake flask culture. In the present study, optimal medium and submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including potato starch 2% (w/v), sucrose 2.5%, soybean 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K 2HPO 4 0.1%, MgSO 4·7H 2O 0.05%, pH 7.0, inoculum size 5%, medium capacity 50 ml/250 ml flask, dispersant 15 beads, culture time 7 days were employed. In fermentation medium, sucrose, beef extract and yeast extract were replaced with molasses of sucrose, groundnut and Vitamin B complex, respectively. Under optimal culture conditions, the yield of polysaccharides production was 9.51 g l −1 after 54 h of fermentation in a 25 l fermenter, which was approximately twice as high as that in shake flask cultures. In addition the entire period of fermentation was shorted to around 1/4 of flask culture time (9 days). Thus, it will meet closely the requirements of industrial fermentation scale of polysaccharides production in C. pruinosa.

Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing NF-κB activation

Cordyceps pruinosa has been used in traditional folk medicine to treat numerous diseases. The molecular mechanism of C. pruinosa pharmacological and biochemical actions of macrophages in inflammation has not been clearly elucidated. We examined how the methanol extract of C. pruinosa regulates production of IL-1β, TNF-α, nitric oxide (NO), and prostaglandin E 2 (PGE 2) in vitro and in vivo. The extract inhibits these inflammatory mediators in LPS-stimulated murine macrophage cell line RAW264.7 and primary macrophages, by suppressing gene expression of IL-1β, TNF-α, inducible nitric oxide synthase, and cyclooxygenase-2. Moreover, the extract suppresses the nuclear transcription factor NF-κB activation in LPS-stimulated RAW264.7 cells. Administration of the extract significantly decreases the plasma levels of these inflammatory mediators in LPS-injected mice. These results suggest that the C. pruinosa methanol extract suppresses inflammation through suppression of NF-κB-dependent inflammatory gene expression, suggesting that the C. pruinosa extract may be beneficial for treatment of endotoxin shock or sepsis.