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OBJECTIVE: To research for the identification of Dongchongxiacao [Cordyceps sinensis( Berk.) Sacc.] and its counterfeits-processed materials from Cordyceps taii Liang et Liu. METHODSE: On the basis of the specimens of Dongchongxiacao and four types of counterfeits, the macroscopic and microscopic identification methods were studied, including the comparison of the macroscopic features, such as insertion position of stroma, colour of caterpillar part, abdominal leg, and the microscopic features such as the transverse section of the stroma, the caterpillar's body wall and the planta of abdominal leg. RESULTS: The differences between Dongchongxiacao and four types of counterfeits were obvious in macroscopic and microscopic features. CONCLUSION: Dongchongxiacao and its counterfeits-processed materials from Cordyceps taii Liang et Liu. can be identified by the differernces of macroscopical and microscopical fetures.
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Objective To investigate the effects of cultivated Cordyceps sinensis on inhibiting the proliferation and migration of B16 melanoma cells. Methods MTT assay and clone formation assay were used to examine the inhibitory effect of cultivated C. sinensis on the proliferation of B16 cells; Scratching test and transwell assay were used to detect its effect on migration; Cell adhesion assay was used to detect its effect on adhesion. Flow cytometry was used to detect its effect on cell cycle arrest; Western blotting was used to detect its effect on the expression of the proteins of MMP-2, MMP-9, Bax, Bcl-2, CyclinD1, CDK2, CDK4, P21, and p-Akt. Results The results showed that cultivated C. sinensis can significantly inhibit the proliferation and clone formation of B16 cells via arresting cells at G1/S phase in a dose-dependent manner. Moreover, cell migration was also substantially inhibited in a dose-dependent manner. Western blotting analysis showed that cultivated C. sinensis obviously increased the levels of Bax and P21, and meanwhile, decreased the expression of Bcl-2, MMP-2, MMP-9, CDK2, CDK4, CyclinD1, and p-Akt. Conclusion The inhibitory effects of cultivated C. Sinensis on the proliferation and migration of B16 cells probably associated with the expression of related regulating proteins, MMPs family proteins and p-Akt protein in cell cycle.
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Objective To establish an HPLC method for simultaneous determination of uridine, vernine, adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine in Cordyceps (Cordyceps militaris, Cordyceps Fungus Powder CS-4, Hirsutella sinensis, and C. sinensis), and determine the characteristic components of C. militaris to provide the scientific basis for quality control of C. militaris and its extract. Methods HPLC was performed on Inertsil ODS-3 column (250 mm × 4.6 mm, 5 μm) with mobile phase A (acetonitrile) and B (water) for gradient elution. The flow rate was 0.8 mL/min, detection wavelength was 260 nm, and column temperature was 30 ℃. Results All mentioned five nucleosides can be detected from C. militaris. However, cordycepin and N6-(2-hydroxyethyl)-adenosine were undetected in the other three Cordyceps species. The sample preparation method of C. militaris has a great effect on the content of nucleosides. The content of uridine, vernine and adenosine was the highest in samples prepared by ultrasonic extraction 180 min. Cordycepin was stable form six sample preparation methods, and N6-(2-hydroxyethyl)-adenosine was unbearable to heat and acid. The contents of cordycepin and N6-(2-hydroxyethyl)-adenosine were the same in four preparation methods. Conclusion This experiment provides a basis for the quality analysis of C. militaris and its extracts. Cordycepin and N6-(2-hydroxyethyl)-adenosine can be used as markers for the quality control of C. militaris and its extracts.
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PA-824 is a novel bicyclic nitroimidazole anti-tuberculosis (TB) drug. Cordyceps sinensis (Berk.) Sacc. (CS) was proven to be a good immunomodulatory compound. This research aimed to investigate the effect of CS on PA-824 in Mycobacterium tuberculosis (M.tb) infected mice (female CBA/J mice, 6 to 8 weeks of age and 20±2 g of weight). Mice were randomly assigned to 4 groups: PA-824, CS, PA-824+CS, and control. To verify the effect of PA-824 and CS on M.tb, after drug administration, mice lungs were harvested and bacterial colony formations were measured. Cells were isolated from infected lungs and spleens to analyze the percentage of CD4+ T cells (CD11a positive). Lung cells were cultured to detect the secretion of interferon-γ (IFN-γ) and interleukin-10 (IL-10) by ELISA. IFN-γ and IL-10 double-positive CD4+ cells in peripheral blood were measured by flow cytometry. The expression levels of IL-2 and IL-10 in mice lungs were analyzed by real-time PCR and western blot. Results showed that PA-824 combined with CS led to the lowest lung colony-forming units (CFU) counts among treated groups. Furthermore, this beneficial outcome might be associated with the decreased CD11a on CD4+ cells in mice lungs and spleens. Moreover, the suppressed secretion of IFN-γ and IL-10, and IL-10 expressions, as well as the decreased IFN-γ and IL-10 double-positive CD4+ cells in blood, could also be associated with the positive effect. However, no significant effect on IL-2 production was found. The combination of PA-824 and CS had more effective bacteriostatic and immunomodulatory effects on M.tb infected mice than PA-824 alone. In conclusion, CS has the potential to be an effective adjuvant in TB treatment.
Subject(s)
Animals , Male , Mice , Anti-Bacterial Agents/pharmacology , Cordyceps/chemistry , Interleukin-10/immunology , Mycobacterium tuberculosis/immunology , Nitroimidazoles/pharmacology , Blotting, Western , Disease Models, Animal , Flow Cytometry , Immunomodulation/drug effects , Immunomodulation/immunology , Mice, Inbred CBA , Mycobacterium tuberculosis/drug effects , Real-Time Polymerase Chain Reaction , Tuberculosis, Pulmonary/immunologyABSTRACT
Objective: To establish an HPLC fingerprint method for water-soluble components in Cordyceps sinensis which could provide the basis for authenticity identification and quality evaluation of C. sinensis. Methods: HPLC method was adopted to construct the fingerprint of water soluble components in C. sinensis and its main confused species (C. liangshanensis, C. gunnii and artificial C. militaris). Total 16 common peaks were marked, and 12 common peaks were confirmed by the method with tandem high performance liquid chromatography (HPLC) and quadrupole time-of-flight mass spectrometry (Q-TOF MS). Results: The common mode of HPLC fingerprint for C. sinensis was developed. The method could distinguish between C. sinensis and its confused species, and 12 common peaks were identified, including cytosine, uracil, cytidine, hypoxanthine + guanine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine, and cordycepin respectively. Conclusion: This method is simple and easy, providing scientific basis for identification of C. sinensis.
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Objective: To investigate the effects of three active components in Cordyceps sinensis (cordycepin, adenosine, and ergosterol) on angiogenesis and the function of hepatic endothelial cells. Methods: Chick chorioallantoic membrane was used to record angiogenesis area, and transgenic zebrafish was used to evaluate the changes of intersegmental vascular and alkaline phosphatase. The toxicity of three active components was assayed in SK-HEP-1 cells by MTT. The proliferation of SK-HEP-1 cells was induced by vascular endothelial growth factor (VEGF), with sorafenib as the positive control. Cell proliferation was analyzed by MTT assay. Cell migration and tube formation were observed by Matrigel and Transwell assay, respectively. Fluorescence probe method was used to detect the levels of intracellular nitric oxide (NO) and nitric oxide synthase (NOS). Results: Adenosine and ergosterol inhibited angiogenesis of chick chorioallantoic membrane, and decreased the number of transgenic zebrafish intersegmental vascular. However, cordycepin can promote angiogenesis of chick chorioallantoic membrane. Compared with the blank control group, VEGF induced SK-HEP-1 cells proliferation, promoted cells migration and tube formation, further significantly increased the levels of NO and NOS in SK-HEP-1. All three active components inhibited the proliferation, tube formation of SK-HEP-1 and decreased the levels of NO and NOS in a dose-dependent manner. Moreover, adenosine and ergosterol inhibited SK-HEP-1 cells migration significantly. Conclusion: Three active components, especially adenosine, could inhibit SK-HEP-1 cells proliferation, migration and tube formation in different degrees. And the mechanism is related to the inhibition of intracellular NO levels and NOS activity.
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Objective: To study the optimum particle size of Cordyceps sinensis for liver fibrosis in combination with in vitro dissolution experiment from serum pharmacology. Methods: To prepare the powder samples with different grinding degrees, Cordyceps sinensis was crushed through 100-, 150-, 200-, and 300-mesh sieves. The in vitro dissolution of adenosine was measured at different time points. Meanwhile, the powder sample was ig administered to rats, and pharmacodynamic approach was adopted to study the inhibition of medicated serum on HSC-T6 proliferation. Results: The accumulative in vitro dissolution of C. sinensis by 200-300 meshes was higher than that of other meshes. Medicated serum could significantly inhibit HSC-T6 cell proliferation. The AUC of HSC-T6 inhibition kinetics of medicated serum crushed to 200-300 meshes was significantly higher than that in other groups. Conclusion: The in vitro dissolution and pharmacodynamic method could be used for the study on different particle sizes of C. sinensis for anti-hepatic fibrosis, and 200-300 meshes are the optimal particle size.
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Objective: The method of loop-mediated isothermal amplification (LAMP) was employed to detect and identify Cordyceps sinensis rapidly. Methods: Specific LAMP primers were designed according to the CS2 serine protease (csp2) gene of Cordyceps sinensis. C. sinensis DNA was extracted using CTAB method. The reaction conditions of LAMP were optimized. Specificity of LAMP reaction was validated by six different strains and using restriction enzyme Taq1 digested the LAMP products. Sensitivity of LAMP was tested with diluted C. sinensis solution with 10-fold gradient. LAMP products were shown by gel electrophoresis or adding SYBR Green I. Results: The method of LAMP for detecting C. sinensis was effective and specific. The detection limit of LAMP assay was up to 6 pg/mL. Conclusion: LAMP protocol is a promising method for the identification and detection of C. sinensis and Chinese materia medica as well.
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Objective To compare the antitumor effects and chemical components of the extracts in cultivated Cordyceps sinensis (Cs) fungus HK-1 and natural Cs. Methods The cultivated Cs fungus HK-1 and natural Cs were extracted with petroleum ether (PE), ethyl acetate (EtOAc), ethanol (EtOH), and water. The cytotoxicity of all the extracts was observed with MTT assay on breast cancer cell line MCF-7, mouse melanoma cell line B16, human premyelocytic leukemia cell line HL-60, and human hepatocellular carcinoma cell line Hep G2. The antitumor activity in vivo of the extract was further studied with animal model. The chemical constituents in the extracts were analyzed by HPLC. Results All of the extracts from the cultivated Cs fungus HK-1 had much stronger cytotoxicity on B16 than those from natural Cs, and the EtOAc extract had the most potent activity. The activity of EtOAc extract from the cultivated Cs fungus HK-1 was more potent than that from natural Cs on all the four cancer cell lines. EtOAc extract from the cultivated Cs fungus HK-1 also can inhibit the growth of tumor in vivo. Chemical components of all extracts were analyzed and ergosterol and adenosine were found to be potential compounds. Conclusion The cultivated Cs fungus HK-1 has stronger antitumor activity than natural Cs and is a potential source of antitumor compounds.
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Object To study the growth speed and dynamic spore production of CY-8202, which is an asexual strain of entomopathogenic fungi isloated from Cordyceps sinensis (Berk.) Sacc., to explore the method of artificial culture of CY-8208 strain, to assay its antibiotic activity and spectrem and to provide the experimental basis for studies of its active components. Methods Variations in colony diameter of the cordyceps hypha cultured on the fungi media were measured. The spore-count was used to determine the dynamic colony spore outputs of the hypha on several fungi media and water agar. The agar-piece method was used to test its antibiotic activity.Results There were linear relationships between the colony extensions and the culture times on common fungi media such as PDA medium, etc.. The amounts of spore produced by the single colony of the fungi were more than 10 7 and gradually increased, but the rates of increase tended to be gentle after 200 h. Antimicrobial tests against 22 microbial strains showed strong inhibition of gram positve bacteria including Bacillus subtilis, Micrococcus tetragenus and Staphylococcus albus, and to gram negative bacteria, including Proteus vulgaris, Salmonella typhi, Aerobacter aerogenes and Salmonella sp., as well as weak inhibition of three mold strains and one actinomycetes strain, but no inhibition was observed in four yeast tested.Conclusion The growth activity and the spore-production ability of Cordyceps hypha are two important factors to infect validly its insect-host. For the growth activity, its strong penetration seems more important than its fast growth. The ability to product numerous spores of Cordyceps hypha may be an important mark of its strong infectivity to insect-host. The antimicrobial tests show that CY-8202 may secrete some metabolites which have a more broad-spectrum antibacterial activity than cordycepin isolated initially from Cordyceps militaris.