Simple HPLC-UV determination of nucleosides and its application to the authentication of Cordyceps and its allies

A simple HPLC-UV method combined with a simple extraction procedure of nucleosides (adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine) was developed and applied to the authentication of Cordyceps and its allies. The separation was performed on a C(18) column by isocratic elution with acetonitrile-water, and UV detection at 260 nm. The amounts of adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine in Cordyceps were 0.28-14.15, 0.006-6.36, 0.01-0.14, 0.68-14.79 and 0.19-20.29 mg/g, respectively. Among the nucleosides studied, cordycepin was characteristically included in Cordyceps militaris (L.) Link. (CM), which is one of key Cordyceps allies, and might be a good marker for authenticating CM. The ratio of nucleosides to adenosine contents in Cordyceps seemed to be a useful marker for authentication and quality control of Cordyceps.     

Quality evaluation of Cordyceps through simultaneous determination of eleven nucleosides and bases by RP-HPLC

A new RP-HPLC method has been developed for the simultaneous determination of 11 nucleosides and bases, including adenosine, cordycepin, cytidine, guanosine, inosine, thymidine, uridine, cytosine, guanine, thymine, and uracil in Cordyceps. Determination was achieved on a Zorbax 300SB C18 analytical column (4.6 x 250 mm id, 5 mm) using gradient elution with diode-array detection. All calibration curves showed good linearity (r2 > 0.9995) within the test ranges. The developed method was simple, rapid, and accurate, and showed good reproducibility for the quantification of 11 nucleosides and bases in natural and cultured Cordyceps with both intra- and inter-day variations of less than 1.8%. Furthermore, hierarchical clustering analysis based on the typical peaks of adenosine, cordycepin, and inosine in HPLC profiles from the 11 tested samples showed that natural and cultured Cordyceps were in different clusters, which could provide a means of discriminating between Cordyceps of different origins. Thus, adenosine, cordycepin, and inosine could be used as markers for quality control of Cordyceps.     

蛹虫草及其培养残基中腺苷和虫草素含量的快速测定

建立了以氰基正相色谱柱反相条件下快速分析腺苷和虫草素的方法。以微波辅助提取蛹虫草及其培养残基样品,每次提取1.5 min,共提取2次。采用Eclipse XDB-CN色谱柱测定提取液中腺苷和虫草素的含量,以甲醇-水(7:93, v/v)为流动相进行等度洗脱,检测波长260 nm,并考察和分析了影响分离性能的流动相组成和pH值。结果腺苷和虫草素在4.5 min内实现完全分离且无基质干扰。腺苷和虫草素在线性范围内线性关系良好,线性相关系数r2分别为0.9998和0.9995,定量限(以10倍信噪比计)分别为0.21 mg/L(腺苷)和0.083 mg/L(虫草素)。该方法日内和日间精密度的相对标准偏差(RSD)小于2%;腺苷和虫草素的平均加标回收率为93.8%～102.9%, RSD值不大于3.62%(n=5)。本方法简便、快速、准确、成本低,可用于冬虫夏草、蛹虫草子实体、虫草培养残基及虫草制剂中腺苷和虫草素含量的快速测定。     

The Role of Autophagy in Anti-Cancer and Health Promoting Effects of Cordycepin

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis, which has been used as an herbal complementary and alternative medicine with various biological activities. The general anti-cancer mechanisms of cordycepin are regulated by the adenosine A3 receptor, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3β, leading to cell cycle arrest or apoptosis. Notably, cordycepin also induces autophagy to trigger cell death, inhibits tumor metastasis, and modulates the immune system. Since the dysregulation of autophagy is associated with cancers and neuron, immune, and kidney diseases, cordycepin is considered an alternative treatment because of the involvement of cordycepin in autophagic signaling. However, the profound mechanism of autophagy induction by cordycepin has never been reviewed in detail. Therefore, in this article, we reviewed the anti-cancer and health-promoting effects of cordycepin in the neurons, kidneys, and the immune system through diverse mechanisms, including autophagy induction. We also suggest that formulation changes for cordycepin could enhance its bioactivity and bioavailability and lower its toxicity for future applications. A comprehensive understanding of the autophagy mechanism would provide novel mechanistic insight into the anti-cancer and health-promoting effects of cordycepin.     

虫草素的合成研究进展

虫草素是从蛹虫草中分离得到的脱氧腺苷结构类似物,具有抗菌、抗病毒、抗肿瘤、免疫调节等广泛的生物活性.虫草素的来源稀缺、价格昂贵,已有许多报道对虫草素的合成进行了研究.本文对这一领域的研究进展进行综述,以期为相关研究提供有价值的参考.     

Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.     

Simple and Rapid Determination of Cordycepin in Cordyceps militaris Fruiting Bodies by Quantitative Nuclear Magnetic Resonance Spectroscopy

The major compound in Cordyceps militaris (C. militaris), cordycepin, has been known to have a variety of pharmacological properties. The cordycepin concentration in artificially cultivated fruiting bodies of C. militaris was determined using quantitative ¹H nuclear magnetic resonance spectroscopy (NMR). The results were compared with a high performance liquid chromatography (HPLC) method. There were no statistically significant differences in the cordycepin concentration by the two methods. Validation of each method was performed in terms of linearity, limit of detection, limit of quantification, intra- and inter-day precision, repeatability, stability, and accuracy. Better inter-day precision, repeatability, stability, and accuracy were obtained by NMR than by HPLC. These results show that NMR is an alternative to HPLC for the determination of cordycepin in C. militaris fruiting bodies.     

Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells

Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3′-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 μM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.     

Voltammetric determination of boron by using Alizarin Red S

Cordycepin is the main active metabolite of Cordyceps militaris extracts; according to recent studies it has interesting therapeutic activities. A new capillary electrophoresis (CE) procedure with UV detection at 254 nm for determination of cordycepin was developed and optimized. Optimal conditions found were 20 mM sodium borate buffer with 28.6% methanol, pH 9.5, separation voltage 20 kV, hydrodynamic injection time 10 s and temperature 25 °C. Linearity was found over the 20-100 μg/mL concentration ranges of cordycepin. The developed method has been applied for determination of cordycepin in various pharmaceutical products. A comparison was made between CE and a high performance liquid chromatography (HPLC) method. Both of these methods gave comparable results. The shorter analysis time and low running cost are the main advantages of CE method.     

Selenium Enrichment on <Emphasis Type="Italic">Cordyceps militaris</Emphasis> 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 18.0 ppm 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 6.49 mg/100 g. So selenium-enriched cultivation may be a potential way to produce more valuable medicinal food as a substitute for wild C. sinensis.     

Preparative separation of high-purity cordycepin from Cordyceps militaris(L.) Link by high-speed countercurrent chromatography

A high-speed counter-current chromatography (HSCCC) technique in a preparative scale has been applied to separate and purify cordycepin from the extract of Cordyceps militaris(L.) Link by a one-step separation. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-n-butanol-methanol-water (23:80:30:155, v/v/v/v) by eluting the lower mobile phase at a flow rate of 2 ml/min under a revolution speed of 850 rpm. HSCCC separation of 216.2 mg crude sample (contained cordycepin at 44.7% purity after 732 cation-exchange resin clean-up) yielded 64.8 mg cordycepin with purity of 98.9% and 91.7% recovery. Identification of the target compound was performed by UV, IR, MS, (1)H NMR and (13)C NMR.     

A Fast,Sensitive and Validated Technique for Eurycomanone and Cordycepin Quantitation using UPLC‒MS/MS

Herbs and herbal based products have been widely used, but some products might contain no herbal ingredient as claimed in the product label. Therefore, it is crucial to develop a fast, sensitive and reliable method to analyze the herbs and their finished products. The roots of Eurycoma longifolia, and the health supplement of E. longifolia and Cordyceps were used as the sample matrices in this study. Sonication assisted extraction was applied to extract the marker compounds, namely eurycomanone and cordycepin from E. longifolia and Cordyceps, respectively. The presence of the marker compounds was established by high throughput ultra-performance liquid chromatography tandem mass spectrometry (UPLC?MS/MS) using a highly sensitive and selective technique of multiple reaction monitoring (MRM). The positive ion transitions in MRM for eurycomanone and cordycepin are m/z 409 → 391 and m/z 252 → 136, respectively. The test method was validated for its robustness, accuracy, precision, linearity, detection and quantitation limits, as well as estimated for its measurement uncertainty.     

Production of Cordycepin and Mycelia by Submerged Fermentation of Cordyceps militaris in Mixture Natural Culture

The submerged fermentation of Cordyceps militaris for cordycepin production and mycelial growth was investigated in this study. Three natural materials of brown rice paste (BRP), beerwort (B), and soybean meal juice (SMJ) were used for fermentation of C. militaris in shaking flasks. The effects of the ratio of three natural materials on dry mycelium weight (DMW) and on cordycepin yield (CY) were analyzed. D-Optional mixture design was used to optimize the ratio of these materials. Compared with the signal culture, the higher mycelial growth and cordycepin production were obtained in mixture. The analysis of Design Expert 6.0 indicated that BRP, B, and SMJ very significantly influenced (P < 0.001) DMW and CY of C. militaris, respectively. The highest DMW (18.96 g/l) and CY (2.17 mg/g) were both obtained at a ratio of 53:6:42. The experiments’ results indicated that the above mixture of these natural materials by D-optional mixture design can be used as a proper medium for the growth of mycelium and the production of cordycepin.     

Optimization of CEC for simultaneous determination of eleven nucleosides and nucleobases in Cordyceps using central composite design

A CEC method is described for the simultaneous determination of 11 nucleosides and nucleobases including cytosine, uracil, uridine, hypoxanthine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenine, adenosine, and cordycepin in Cordyceps using 5-chlorocytosine arabinoside as internal standard (IS). Chemometric optimization based on central composite design was employed to find the optimum conditions. The factors for optimization were defined as three parameters: voltage, pH, and concentration of ACN as organic modifier. The resolution (R(s)) between inosine and guanosine, as well as the entire run time were employed to evaluate the response function. A running buffer composed of 4 mM ammonium acetate and 2 mM triethylamine (TEA) adjusted to pH 5.3 using acetic acid, and containing 3% ACN as modifier, with gradient voltage (0-4 min: 20 kV, 4-12 min: linear gradient from 20 to 30 kV; 12-16 min: 30 kV) were found to be the optimum conditions for the separation. Separation of the 11 investigated compounds and 5-chlorocytosine arabinoside was achieved within 16 min. The contents of the 11 compounds in natural and cultured Cordyceps sinensis, and cultured Cordyceps militaris were also compared. The result showed that CEC is an efficient method for analysis of nucleosides and nucleobases in Cordyceps, which is helpful to control the quality of this valued traditional Chinese medicine.     

Anti-adipogenic activity of Cordyceps militaris in 3T3-L1 cells

Inhibition of adipocytes differentiation is suggested to be an important strategy for prevention and/or treatment of obesity. In our present study, Cordyceps militaris showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes as assessed by measuring fat accumulation using Oil Red O staining. Activity-guided fractionation led to the isolation of cordycepin (1), guanosine (2) and tryptophan (3) as active compounds. All the three compounds were more effective in the prevention of early stage of adipogenesis than in lipolysis. In addition, combinational treatment of three compounds significantly increased anti-adipogenic activity.     

Functional Analysis of Ribonucleotide Reductase from <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cordyceps militaris produces cordycepin (3′-deoxyadenosine), which has various activities, including anti-oxidant, anti-tumoral, anti-viral, and anti-inflammatory. Ribonucleotide reductase (RNR) seems to be a candidate to produce cordycepin in C. militaris because RNR catalyzes the reduction of nucleotides to 2′-deoxynucleotides, whose structures are similar to that of cordycepin. However, the role of RNR has not been confirmed yet. In this study, complementary DNAs (cDNAs) of C. militaris RNR (CmRNR) large and small subunits (CmR1 and CmR2) were cloned from C. militaris NBRC9787 to investigate the function of CmRNR for its cordycepin production. C. militaris NBRC9787 began to produce cordycepin when grown in a liquid surface culture in medium composed of glucose and yeast extract for 15 days. CmR1 cDNA and CmR2 cDNA were obtained from its genomic DNA and from total RNA extracted from its mycelia after cultivation for 21 days, respectively. Recombinant CmR1 and CmR2 were expressed individually in Escherichia coli and purified. Purified recombinant CmR1 and CmR2 showed RNR activity toward adenosine diphosphate (ADP) only when two subunits were mixed but only show the reduction of ADP to 2′-deoxyADP. These results indicate that the pathway from ADP to 3′deoxyADP via CmRNR does not exist in C. militaris and cordycepin production in C. militaris may be mediated by other enzymes.     

Effects of Selenium and Light Wavelengths on Liquid Culture of <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Link

To investigate the effects of selenium and light wavelengths on the growth of liquid-cultured Cordyceps militaris and the main active components’ accumulation, culture conditions as selenium selenite concentrations and light of different wavelengths were studied. The results are: adenosine accumulation proved to be significantly selenium dependent (R ² = 0.9403) and cordycepin contents were determined to be not significantly selenium dependent (R ² = 0.3845) but significantly enhanced by selenium except for 20 ppm; there were significant differences in cordycepin contents, adenosine contents, and mycelium growth caused by light wavelengths: cordycepin, blue light > pink light > daylight, darkness, red light; adenosine, red light > pink light, darkness, daylight, blue light; and mycelium growth, red light > pink light, darkness, daylight > blue light. In conclusion, light wavelength had a significant influence on production of mycelia, adenosine, and cordycepin, so lightening wavelength should be changed according to target products in the liquid culture of C. militaris.     

The correlation between molecular weight and antitumor activity of galactosaminoglycan (CO-N) from Cordyceps ophioglossoides

A galactosaminoglycan (CO-N) obtained by ultrasonication from a protein-bound polysaccharide SN-C, which was isolated from Cordyceps ophioglossoides culture, has a direct cytotoxicity against tumor cells (Ohmori et al., Chem. Pharm. Bull., 37, 1019 (1989). High performance liquid chromatographic analysis revealed that CO-N shows a broad molecular weight distribution with an average molecular weight of 33000. A potent antitumor activity of CO-N was observed in the higher-molecular-weight fraction on gel filtration, and the low-molecular-weight fraction below 6600 showed a weak activity. However, the depolymerized CO-N (ca. 5500) obtained by further ultrasonication of the original CO-N still retained the antitumor activity of CO-N against Ehrlich ascitic carcinoma or MM46 solid mammary carcinoma.     

Simultaneous determination of cordycepin and its metabolite 3′-deoxyinosine in rat whole blood by ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry and its application to accurate pharmacokinetic studies

Cordycepin from Cordyceps possesses excellent pharmacological properties, including anti-inflammation and anti-tumor effects, therefore representing a potential alternative medicine. However, doubts about the pharmacokinetic results of cordycepin had been raised in the previous study due to its rapid deamination. The organic solvent methanol was immediately added to terminate the degradation of cordycepin in anticoagulated blood samples and enable the accurate evaluation of pharmacokinetics in vivo. A sensitive and selective ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry method was developed and validated to simultaneously determine cordycepin and its deamination metabolite 3′-deoxyinosine using 2-chloroadenosine as an internal standard in rat whole blood. The calibration curves of cordycepin and 3′-deoxyinosine showed excellent linearity within the concentration range of 1.05–10 000.00 ng/ml with acceptable accuracy, precision, selectivity, recovery, matrix effect, and stability. This method was successfully applied to the pharmacokinetic study of cordycepin and its metabolite in rat blood. The effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride on the pharmacokinetics of cordycepin was investigated. In summary, the reliable pharmacokinetic parameters of cordycepin and its deamination metabolite 3′-deoxyinosine in rat blood were successfully elucidated. Erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride considerably prolonged the half-life of cordycepin in vivo.     

Cordycepin inhibits UVB-induced matrix metalloproteinase expression by suppressing the NF-��B pathway in human dermal fibroblasts

Cordycepin (3''-deoxyadenosine) has been shown to exhibit many pharmacological activities, including anti-cancer, anti-inflammatory, and anti-infection activities. However, the anti-skin photoaging effects of cordycepin have not yet been reported. In the present study, we investigated the inhibitory effects of cordycepin on matrix metalloproteinase-1 (MMP-1) and -3 expressions of the human dermal fibroblast cells. Western blot analysis and real-time PCR revealed cordycepin inhibited UVB-induced MMP-1 and -3 expressions in a dose-dependent manner. UVB strongly activated NF-κB activity, which was determined by IκBα degradation, nuclear localization of p50 and p65 subunit, and NF-κB binding activity. However, UVB-induced NF-κB activation and MMP expression were completely blocked by cordycepin pretreatment. These findings suggest that cordycepin could prevent UVB-induced MMPs expressions through inhibition of NF-κB activation. In conclusion, cordycepin might be used as a potential agent for the prevention and treatment of skin photoaging.