Paecilomyces Hepiali Research Articles

Free-Radical Scavenging Activities of Cultured Mycelia of Paecilomyces hepiali (Ascomycetes) Extracts and Structural Characterization of Bioactive Components by Nuclear Magnetic Resonance Spectroscopy.

Current research is focused on testing the cultivation of Paecilomyces hepiali mycelia on various plant substrates and producing fungus or mycelial biomass with qualitatively interesting substances. P. hepiali mycelia was cultivated using solid-state fermentation of different substrates. Mycelial biomass was then analyzed, and antioxidant activity was evaluated using the DPPH radical scavenging method for different ethanolic extracts based on a millet substrate (extract 1) or a chickpea substrate (extract 2). Extract 1 corresponds to a half-maximal DPPH radical inhibitory concentration of 1.73 mg/mL; the inhibitory concentration of ethanol extract 2 was almost 4.5 times higher at 7.92 mg/mL. Extracts 1 and 2 were separated into fractions by column chromatography and the chemical structures were determined for the substances that formed the most effective fraction of sample 1. The chemical structures of all compounds in the most active fraction of sample 1 were analyzed by 1H, 13C, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear single-quantum correlation spectroscopy, and heteronuclear multiple-bond correlation spectra.

Investigations on the antifatigue and antihypoxic effects of Paecilomyces hepiali extract.

Paecilomyces hepiali, one of the most valuable and effective Chinese medicinal herbs, possesses potential antioxidant, immunomodulatory, antitumor and anti‑inflammatory properties. The present study aimed to investigate the antifatigue and antihypoxic effects of Paecilomyces hepiali extract (PHC) in a mouse model. Using a rotating rod, forced swimming and running assessment, the antifatigue activity of PHC was determined. PHC administration for 7 days had no effect on mouse horizontal or vertical movement, indicating no neurotoxicity at the selected doses was observed. Using a normobaric hypoxia, sodium nitrite toxicosis and acute cerebral ischemia assessments, PHC was confirmed to possess antihypoxic effects. PHC treatment for 7 days significantly enhanced the serum and liver levels of adenosine triphosphate, superoxide dismutase and glutathione peroxidase, prior to and following 60 min of swimming. The levels of antioxidant‑associated proteins in the livers of the mice were analyzed using western blotting. PHC effectively increased the expression levels of phosphorylated (p)‑5'‑monophosphate (AMP)‑activated protein kinase (AMPK), p‑protein kinase B (AKT) and p‑mammalian target of rapamycin (mTOR). The results of the present study demonstrated that PHC efficiently enhanced endurance from fatigue and had antihypoxic effects through elevation of the antioxidant capacity in the serum and liver, at least in part through the AMPK and AKT/mTOR pathways. These results indicate the potential of this natural product as an antioxidant in the treatment of fatigue, hypoxia and their associated diseases.

Anti-inflammatory effects of Cordyceps mycelium (Paecilomyces hepiali, CBG-CS-2) in Raw264.7 murine macrophages.

Cordyceps (CS) is a traditional Chinese herb with various biological effects that include immune modulation. CBG-CS-2 is a strain, Paecilomyces hepiali, of the Cordyceps spp. The anti-inflammatory effects of CBG-CS-2 were investigated. The water-soluble fraction of CBG-CS-2 has high anti-inflammatory activity in LPS-induced Raw264.7 macrophages. We tested the role of CBG-CS-2 on the anti-inflammation cascade in LPS-stimulated Raw264.7 cells. CBG-CS-2 significantly decreased NO production, iNOS expression, and pro-inflammatory cytokine secretion in a dose-dependent manner. To investigate the mechanism by which CBG-CS-2 inhibits NO, iNOS, and pro-inflammatory cytokines, we examined the activities of NF-κB and AP-1 in LPS-activated macrophages. The results demonstrate that CBG-CS-2 suppresses the production and expression of NO, iNOS, and pro-inflammatory cytokines in LPS-activated macrophages via inhibition of NF-κB and AP-1, which may play an important role in inflammation. These findings suggest that CBG-CS-2 has modulatory effects on the inflammatory system in macrophages, and that it can serve as a useful anti-inflammatory dietary supplement or drug.

Altered proteomic polymorphisms in the caterpillar body and stroma of natural Cordyceps sinensis during maturation.

ObjectiveTo examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem.MethodsThe surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%–4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster.ConclusionProteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.

A Review on Fungal Isolates Reported as Anamorphs of Ophiocordyceps sinensis

This brief review presents current developments on Ophiocordyceps sinensis and fungal strains which have been reported as its anamorphs. A survey of literature has shown that Hirsutella sinensis is currently receiving general acceptance as a true anamorph of O. sinensis. This isolate has been confirmed as the true anamorph by both morphological and molecular methods. The other isolates such as Paecilomyces sinensis, Scytalidium hepiali, Tolypocladium sinensis, Chrysosporium sinensis, Synnematium sinensis, Paecilomyces hepiali, Mortierella hepiali, and Scytalidium hepiali have been discarded as anamorphs of O. sinensis. The review also discusses various methods used to determine or confirm anamorphs of O. sinensis. Considering that the methods have strengths and weaknesses of varying magnitudes, a collective use of various methods is recommended for more reliable conclusions.

Influences of carbon sources on the biomass, production and compositions of exopolysaccharides from Paecilomyces hepiali HN1

The parasitic fungus, Paecilomyces hepiali, is used to produce Cordyceps materials as succedaneum of natural Cordyceps sinensis in China. The purpose of this research was to investigate the effects of glucose, mannose, sucrose, lactose as solo carbon source and sucrose + lactose or mannose + sucrose as synthetic carbon source on the growth of mycelium and production, chemical composition, molecular weight distribution and monosaccharide composition of exopolysaccharides from P. hepiali HN1 (PHEPS). The maximum mycelium biomass of 12.16 kg m−3 and PHEPS yield of 4.57 kg m−3 were achieved from the culture with sucrose (50 kg m−3) as carbon source. The resulting PHEPS was characterized by analyses of chemical composition, size-exclusion chromatography and high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization. It was found that the chemical compositions and monosaccharide ratios in PHEPS were significantly affected by the carbon sources used. Glucose or mannose as carbon source enhanced the biosynthesis of PHEPS with higher-molecular weight (>1000 kD), but solo carbon source of lactose or synthetic carbon source of mannose + lactose did not increase the ratio of galactose in PHEPS. The metabolism kinetics of carbon sources demonstrated the correlation between PHEPS synthesis and the utilization of carbon sources. These findings will be useful for further works on the production, structure and function of PHEPS.

Nutrigenomic Study on Immunomodulatory Function of <i>Cordyceps</i> Mycelium Extract (<i>Paecilomyces hepiali</i>) in Mitomycin C–Treated Mice

Cordyceps (CS) is a Chinese herb that produces various effects including immune modulation, and now CS culture product is interested in the use as a functional food. We prepared CS mycelium culture extract (Paecilomyces hepiali, CBG-CS-2), CS extract, for functional foods. This study aimed to deduce the molecular mechanism of immunomodulatory effect of CS extract in Peyer’s patches (PPs), a main gut immune site, in mice that treated with mitomycin C (MMC), an immunosuppressing antibiotics. Nutrigenomics give us invaluable molecular information about both of foods and nutrition to improve or maintain good health. Here we performed nutrigenomics using DNA microarray to investigate the effect of CS extract on gene expression altered in PPs of the immunosuppressed mice. Interestingly, CS extract protected from the MMC-mediated downregulation of 22 genes, which are associated with IgA production and other immune response in PPs. These suggested that CS extract alleviated the downregulated expression of immune related genes in the gut immune site in an immunosuppressed state. Thus CS extract appears to be practical functional food for immunodepression and/or its related hypofunction.

A Comparative Study on Chemical Composition and Pharmacological Effects of <i>Paecilomyces hepiali</i> and Wild <i>Ophiocordyceps sinensis</i>

This study looked at comparison of chemical components and pharmacological activity between wild Ophiocordyceps sinensis and Paecilomyces hepiali. The chemical components investigated included amino acids, vitamins, dietary elements, protein, lipid, ash, carbohydrates, crude fibre, ergosterol and mannitol. Studies on pharmacological activity included anti-platelet aggregation, inhibitory effect on IL-8 gene expression, anti-mutagenic activity, skin whitening effect and impro- vement activity on human skin texture. The results show that P. hepiali has a larger total content of seven essential amino acids (leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine and valine) than O. sinensis, 8580 mg/100g and 6180 mg/100g respectively. The total content of dietary elements analysed (potassium, magnesium, zinc, copper, manganese and selenium) was also higher in P. hepiali (3135 mg/100g) than that in O. sinensis (2445 mg/100g). The total content of four vitamins (B1, B2, B6 and E) was almost equal for both fungi. Paecilomyces hepiali had more content of protein, lipid, ash, carbohydrate, ergosterol and mannitol than O. sinensis. However, the contents of lipid and ash were not significantly different between the two fungi. The hot water extract of P. hepiali has been shown to have a number of pharmacological effects which include 1) inhibition of aggregation on human platelet, 2) inhibition of IL-8 gene expression, 3) anti-mutage- nic activity, 4) inhibition effect on production of melanin, and 5) improvement on human skin texture. Anti-platelet aggregation effects on human platelet, inhibition of IL-8 gene expression, anti-mutagenic activity and improvement effect on human skin texture were greater in P. hepiali treatments than that in wild O. sinensis.

Effects of hot-water extract of <i>Paecilomyces hepiali</i> on hypertension parameters in Spontaneously Hypertensive Rats

In this study, effects of hot water extract of Paecilomyces hepiali mycelia on hypertension parameters in spontaneously hypertensive rats (SHR) were investigated. The tested parameters included blood pressure, blood and urine biochemical composition, renin and angiotensin II levels in the blood. Prior to these tests, the extract was examined for toxicity. The fungus was cultivated in a solid medium composed of 40 g brown rice, 0.32 g glucose, 0.65 g sucrose, 2 g peptone and 65 ml corn steep liquor. No abnormality or harmful effects were observed in the toxicity test. Administration of a continuous-dose, once daily, to SHR for 27 weeks (from 13 weeks of age) decreased the systolic blood pressure (SBP) significantly. Levels of blood urea nitrogen, β-lipoprotein lipid peroxides and low density lipoprotein were significantly lower in the treated groups when compared to the control group. Urinary protein was significantly reduced in the middle and high dose groups. In comparison with the control group (0 mg/kg/10ml/day), significantly higher values were obtained for total cholesterol in groups that were given middle (170 mg/kg/10ml/day) and high (250 mg/kg/10ml/day) dosages. In all dosages (low, middle and high) the values for triglyceride were significantly higher than value found in the control group. In terms of angiotensin II levels, the value in the control group was markedly higher than values in the other groups. The results suggest that oral administration of hot water extract of P. hepiali mycelia has ability to control hypertension in rats.

Optimization for production of exopolysaccharides with antitumor activity in vitro from Paecilomyces hepiali

In the present study, optimal medium for the growth of mycelia and the production of exopolysaccharides from Paecilomyces hepiali HN1 (PHEPS) in submerged culture was investigated. As a result, the maximum production of mycelia (12.98±0.14g/L) and PHEPS (5.33±0.11g/L) were achieved under the optimal medium of sucrose 46.08g/L, yeast extract 4.71g/L, (NH4)2SO4 5.72g/L, KH2PO4 1.70g/L, CaCl2 0.50g/L, MgSO4 0.50g/L, potato extract 1% and malt extract 1%. Furthermore, the antitumor activity of PHEPS in vitro was evaluated by using three cell lines of human liver tumor HepG2 cells, breast cancer MCF-7 cells and cervical cancer Hela cells. It was found that PHEPS exhibited relative higher anti-proliferative activity against HepG2 cells than MCF-7 cells and Hela cells. At a concentration of 500μg/mL and 72h treatment, the inhibition rate of PHEPS on HepG2 cells reached to 62.58%. All these results suggested that PHEPS could be explored as novel natural antitumor agent with great potential application.

Optimization of ultrasonic extraction of mycelial polysaccharides from Paecilomyces hepiali using response surface methodology and its antioxidant activity

Ultrasonic technology was applied to extract mycelial polysaccharides fromPaecilomyces hepiali and the process was optimized by response surface methodology. Antioxidant activity of polysaccharides was also investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (%DPPHsc). Three independent variables were ratio of water to raw material (x1), ultrasonic power (x2) and extraction time (x3). Statistical analysis indicated that x1, x2, x12, x32, x1x2, x1x3and x2x3 had significant effect on the yields. Besides, x2, x12 and x22 shaped the %DPPHsc of polysaccharides significantly. Three dimensional surface plots and contour plots were drawn from the mathematical model. The optimal conditions for yield and %DPPHsc were as follows: condition (1) x1 = 125 mL/g, x2 = 300 W, x3 = 12 min, and condition (2) x1 = 125 mL/g and x2 = 500 W, x3 = 11 min, respectively. Under these conditions, the yield was 9.37%, and %DPPHsc was 45.34%. There existed good agreement between experimental and predicted values. Key words: Ultrasonic extraction, polysaccharides, Paecilomyces hepiali, response surface methodology, antioxidant activity.

Pro-apoptotic effects of Paecilomyces hepiali, a Cordyceps sinensis extract on human lung adenocarcinoma A549 cells in vitro

Paecilomyces hepiali (PH) is a derivative of Cordyceps sinensis (CS), a fungus that has been shown to have anti-cancer and pro-apoptotic effects. Here, we aimed to investigate the effect of in vitro PH treatment on cell proliferation, cell cycling, apoptosis, and tumor necrosis factor-alfa (TNF-α) mRNA expression in human lung adenocarcinoma A549 cells (A549). A549 cells were treated with an aqueous extract of PH at concentrations of 0.25, 0.5, 1, 2, and 4 mg/ml. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cellular viability and proliferation, while flow cytometry (FCM) was used to examine cell cycling. Apoptosis was assayed using Annexin V Fluorescein Isothiocyanate / Propidium Iodide (V-FITC/PI) and FCM. TNF-α mRNA expression was measured with reverse transcriptase-polymerase chain reaction (RT-PCR). Cell proliferation was significantly suppressed by treatment with 1, 2, and 4 mg/ml of PH extract. Furthermore, the MTT assay showed that cell proliferation was inhibited in a concentration-time-dependent manner. As the concentration of the PH treatment increased, there were fewer cells in the S phase, and more cells in the G0/G1 and G2 phases. After 24 h of treatment, apoptosis was induced by a dose of 2 mg/ml of PH. TNF-α mRNA expression was significantly higher in the intervention groups and was positively associated with treatment concentration. These results indicate that in vitro treatment with an aqueous extract from PH limits cell proliferation, induces apoptosis, and causes cell cycle arrest of A549 cells; this suggests that it may have potential as a therapy for lung adenocarcinoma.

Slow and steady phosphate solubilization by a psychrotolerant strain of Paecilomyces hepiali (MTCC 9621)

A psychrotolerant phosphate solubilizing fungus has been isolated from the rock soil of a cold desert site in Indian Himalaya. The fungus grows from 4 to 35°C (optimum 21°C), and from 2 to 13.5 pH (optimum 9) under laboratory conditions. Based on phenotypic characters and 26S rDNA analysis, the fungus is identified as Paecilomyces hepiali. In quantitative estimation that was carried out at 9, 14, and 21°C, the fungus solubilized maximum phosphate at 14°C. In view of the slow growth and persistence of the desired activity at low temperature, the estimation was carried out for a longer period, i.e., up to 6 weeks. The suboptimal conditions for growth and biomass production were found to be optimal for phosphate solubilization by the fungus. At 14 and 9°C, the solubilization touched its maximum on day 42. Decline in pH was found to be significantly correlated with the phosphate solubilization at all the temperatures, under consideration. The acid phosphatase activity was found to be more prominent than alkaline phosphatase in culture filtrate. High performance thin layer chromatography (HPTLC) analysis showed production of six organic acids, gluconic and α-keto glutaric acid being in maximum amount in the culture filtrate. The study has ecological significance in view of the nutrient cycling under low temperature environment, prevalent in Himalayan region.

Maturational Alteration of Oppositely Orientated rDNA and Differential Proliferation of GC- and AT-biased Genotypes of Ophiocordyceps sinensis and Paecilomyces hepiali in Natural Cordyceps sinensis

Multiple reports have detailed the simultaneous detection of ribosomal DNA (rDNA) of Paecilomyces hepiali (Ph), Hirsutella sinensis and two genotypes of Ophiocordyceps sinensis (Os) from Cordyceps sinensis (Cs), and the altered phenotypes and chemical components of Cs during Cs maturation. In this study, we observed an increase in the biomass of Ph and two moieties of Os rDNA during Cs maturation. The GCand AT-biased genotypes of transition mutations in the Cs stroma were confirmed using MassARRAY SNP genotyping. In premature Cs, the AT-biased genotypes were not expressed in the caterpillar, but highly predominated in the stroma. The GC bias was expressed in an opposite manner in the premature Cs compartments. The differential expression of GC- and AT-biased mutants was altered during Cs maturation. The increased biomass of the inverse-oriented Os rDNA of the GC-genotype was associated with a maturational increase in stroma height. We also report evidence of transversion mutations within Cs genes. The dynamic alterations of Ph and Os mutant gene expressions along with Cs maturation may play an essential role in Cs germination and maturation, the key elements of Cs life cycle, and result in the varied therapeutic potency of Cs.

Maturational alteration of differential expressions of GC:AT‐biased genotypes of Cordyceps sinensis fungi and Paecilomyces hepiali in Cordyceps sinensis

We reported simultaneous detections of the rDNA of Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs) in Cordyceps sinensis (Cs), a precious Chinese herb, and the altered phenotypes of Cs mutants with Cs maturation. In this study we performed Southern blotting analysis and found dramatic maturational increases in the biomasses of Ph and Hs rDNA. Two moieties are shown on the Hs blot when the genomic Cs DNA was prepared with EcoRI. MassARRAY SNP genotyping revealed both GC‐ and AT‐biased mutants in Cs stroma. The AT‐biases did not express in premature Cs caterpillar, but highly predominately in premature stroma. The GC‐bias expresses oppositely in the premature Cs compartments. The differential expressions were altered non‐proportionally with Cs maturation. The expression of the AT‐biases was similar to that for Ph. When Cs stroma genomic DNA was used as the template, inversely orientated rDNA and EcoRI‐digestible PCR products within ITS regions were increased proportionally to the increases in stroma height, indicating Cs maturation‐related DNA mutations and reverse fusion of chromosomes. In conclusion, Cs maturation associates with augmented biomasses of both Ph and Cs mutant genes and altered differential expressions of Cs fungi with GC:AT‐biased genotypes, representing an element in Cs lifecycle important to the anamorph‐telemorph connection of this natural herb.

Transition mutations of Hirsutella sinensis genes and expressions of Paecilomyces hepiali genes during germination and early development of Cordyceps sinensis stroma

We reported simultaneous detections of 2 live fungi, Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs), and their genes in Cordyceps sinensis (Cs) (J Mycol Res 5:214, 2007). With Cs maturation, Hs displayed a >50% decline of its competitive colony forming ability (p<0.001), and profiles of component proteins and small organic chemicals were altered. In this study with use of Ph (ITS 5‐179) and Hs (ITS 5‐162) specific probes, Southern blotting revealed large increases in expressions of Ph and Hs genes with Cs maturation. Two species of Hs‐related Cs genes (87% identity) were identified, consisting mainly of C‐T and G‐A mutations. In premature Cs, the GC genotype expressed only in caterpillar body, while mainly AT genotype in stroma. The Ph genes and the AT genotype Hs genes appear to express synchronously during germination and early development of Cs stroma. When Cs matures, the AT genotype starts to express in caterpillar and the GC genotype in stroma. It is assumed that essential enzymes needed for the transitions between GC and AT genotypes may be provided by co‐existing fungus Ph and play key roles in assisting the anamorph‐telemorph connection of Cs.

β Cell Protecting and Immunomodulatory Activities of Paecilomyces Hepiali Chen Mycelium in STZ Induced T1DM Mice

The anti-hyperglycemic and immunomodulatory activities of the ethanol extract from Paecilomyces Hepiali Chen (PHC), a Chinese medicine, were investigated in streptozotocin-induced type 1 diabetic (T1DM) mice. Male Balb/c mice, which were i.p. injected with streptozotocin (STZ, 50 mg/kg, for 5 consecutive days) on Day 7, were orally administered saline (the normal control and diabetic control group), Metformin (60 mg/kg, b.w., positive group), or the extract (100 mg/kg, b.w., PHC prevention group) from Day 1 to Day 28, Mice i.p. injected with streptozotocin (STZ, 50 mg/kg, b.w.) for 5 consecutive days prior to PHC treatment (100 mg/kg, b.w.) were used as the PHC treatment group. The effects of PHC on postprandial blood glucose concentrations, plasmatic insulin levels, morphology of pancreatic beta cells and CD4(+) T cells proliferation after 28-day treatment were monitored. Results showed that PHC administered 6 days before STZ induction of diabetes in mice significantly decreased blood glucose level (p < 0.01). An increase of insulin level was also observed as compared to those in the diabetic control group (p < 0.01). In addition, fewer inflammatory cells infiltrated the pancreatic islet and fewer beta cells death by apoptosis within the inflamed islets were observed. More importantly, the CD4(+) T cell proliferation was remarkably attenuated ex vivo in mice preventively treated with PHC (p < 0.01). In comparison to the PHC prevention group, no significant hypoglycemia, changes of insulin level and beta cell protection were observed in mice treated with PHC after STZ administration. Our findings demonstrated that preventive administration of PHC protected beta cells from apoptosis in type 1 diabetes induced by STZ, and the underlying mechanism may be involved in suppressing CD4(+) T cells reaction, reducing inflammatory cells infiltration and protecting beta cell apoptosis in pancreatic islet.

Dynamic alterations of chemical component profiles during Cordyceps sinensis maturation and comparisons with those for Hirsutella sinensis and Paecilomyces hepiali mycelia

Cordyceps sinensis (Cs) is a precious Chinese herb traditionally used for anti‐aging and rehabilitation from diseases. Co‐existence of Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs) in Cs has been reported (FESAB 19:A1033, 2005; 21:A1079, 2007). We examined maturational alterations of component proteins and small organic chemicals in fresh Cs (180 pieces) and compared the profiles with those for Ph and Hs. Cs of 2 maturation stages were used, less mature Cs (T1) with a plump caterpillar body (CB) and a short stroma (Stm), and mature Cs (T2) with a less plump CB and a long Stm. Cs (CB and Stm) and freeze‐dried mycelia of Ph and Hs were individually grinded in a mortar into powder in liquid nitrogen and dissolved in water or methanol before subjected to the profiling analysis. SELDI‐TOF MS was used for protein profiling and HPLC with an ODS‐C18 column for small molecule profiling. Different profiles were noted for Cs (CB, Stm) in 2 maturational stages. The profiles for Ph and Hs did not match completely to those for Cs. But Ph mycelia appears to show closer profile patterns to Cs, than Hs mycelia does. In conclusion, Cs maturation associates with dynamic changes in profiles of component chemicals, supporting its superior function profiles. As compared to Hs, the profiles for Ph are closer to those for Cs.

Declines of proliferation predominance of Hirsutella sinensis associate with unparallel alterations of its DNA copies during maturation of Cordyceps sinensis

Cordyceps sinensis (Cs) is a precious tonic herb used in China for centuries. We reported simultaneous detection of 2 live fungi, Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph), and their DNA in fresh Cs (FASEB J 19:1033, 2005; 21:A1079, 2007). A companion study (#1476) indicated a 50% decline of competitive proliferation capability of Hs during Cs maturation. We further examined an association of such a predominance decline with possible alterations of Hs gene copies. Hs and Ph ITS sequences were extended up‐ and downstream to their partial 18S or 28S DNA for designing and producing internal control probes for Southern analysis, with use of 18S‐ and 28S‐specific primers. With use of Hs and Ph specific probes, Southern blotting was performed to examine the gene copies of Hs and Ph in Cs of 2 different maturation stages, less mature Cs (T1) collected in early May and mature Cs (T2) in late June. Southern blotting demonstrated slightly altered amounts of Hs rDNA in mature Cs (caterpillar bodies and stromata) collected in late June, as compared to the less mature counterparts. Our data suggest paradoxical dissociation of the copies of Hs rDNA and competitive proliferation capability of Hs (50% drop) during Cs maturation, indicating other factors possibly accounting for the decline of proliferation dominance of Hs.

Maturational alterations of proliferation predominance of Hirsutella sinensis and Paecilomyces hepiali in natural Cordyceps sinensis

Cordyceps sinensis (Cs) has been used for centuries in China as a medicinal herb for Yin‐Yang double‐invigorations. We reported co‐existence of Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs) in Cs (FESAB 19:A1033, 2005; 21:A1079, 2007). In this study, 206 pieces of fresh Cs were collected in Kangding, Sichuan (Qinghai‐Tibetan Plateau) in mid May (0–10 degree C) and late June (10–20 degree C). Of them, 120 pieces were immature Cs (T1) with a plump caterpillar body (CB) and a short stroma (Stm); and 86 were mature Cs (T2) with a less plump CB and a long Stm. Fresh Cs were washed and surface sterilized in 0.1% Mercuric Chloride for 2–10 min, followed by washing (3×) with sterile water. CB and Stm of 206 pieces of Cs were individually cut into pieces (1.5∼2.0 mm thick), and placed on top of slant PDAP culture medium. Culture was done at 10 degree C. Growth of Hs colonies was found in 82 tubes (39.8%) of Cs tissue slides: 61 (50.8%) of T1 Cs and 21 (24.4%) of T2 Cs (p&lt;0.001). At the unfavorable low culture temperature, Ph was grown in 12 tubes (5.83%) of Cs tissue slides: 5.83% for T1 Cs and 5.81% for T2 (p&gt;0.50). In conclusion, the proliferation predominance of Hs decreases along with Cs maturation, and of Ph needs to be further checked at an optimal temperature for Ph.