Aqueous Extracts of Cordyceps kyushuensis Kob Induce Apoptosis to Exert Anticancer Activity.

Cancer has become the leading cause of mortality since 2010 in China. Despite the remarkable advances in cancer therapy, a low survival rate is still a burden to the society. The antineoplastic activity of aqueous extracts of Cordyceps kyushuensis Kob (AECK) was measured in this study. Results showed that AECK can significantly inhibit the proliferation and viability of U937 and K562 when treated with different concentrations of AECK, and the IC50 values of U937 and K562 were 31.23 µg/ml and 62.5 µg/ml, respectively. Hoechst 33258 staining showed that AECK could cause cell shrinkage, chromatin, condensation, and cytoplasmic blebbing, and DNA ladder experiment revealed the evident feature of DNA fragmentation which showed that AECK could induce cell apoptosis. Moreover, AECK gave rise to intrinsic apoptosis through increasing the amount of Ca2+ and downregulating the expression of Bcl-2. Meanwhile, the level of Fas death receptor was elevated which indicated that AECK could lead to exogenous apoptosis in U937. The expressions of oncogene c-Myc and c-Fos were suppressed which manifested that AECK could negatively regulate the growth, proliferation, and tumorigenesis of U937 cells. This research presented the primary antitumor activity of AECK which would contribute to the widely use of Cordyceps kyushuensis Kob as a functional food and medicine.

[Analysis and evaluation of different radial parts of Cordyceps kyushuensis by Fourier transform infrared spectroscopy].

Fourier-transform infrared spectroscopy (FTIR) and second derivative spectra were used to analyze and evaluate the different parts of Cordyceps kyushuensis Kob in the present work. The results showed that C. kyushuensis contained proteins, polysaccharides, nucleosides, lipids and other active ingredients, the single-dimensional IR spectra of the various parts were highly similar, the similarity coefficient between the cultured stroma and medium reached up to 0.992 7, and the natural stroma was more different from the parasites, with similarity coefficient of 0.949 9. Second derivative spectrum further enriched and confirmed the feature of corresponding spectrum peaks, proved the existence of active substances such as cordycepin and adenosine, and prompted the presence of alpha- and beta-glycosidic bonds. The diversity and complexity of chemical constituents in different parts of C. kyushuensis were synthetically described by IR spectra, which provided a fast, comprehensive and objective approach to the analysis and evaluation of the imperceptible differences, and the quality control of Cordyceps. This work supplies a theoretical basis for development and utilization of genetic resources C. kyushuensis.

Supercritical fluid extraction of quinolizidine alkaloids from Sophora flavescens Ait. and purification by high-speed counter-current chromatography.

Supercritical fluid extraction (SFE) was used to extract quinolizidine alkaloids from Sophora flavescens Ait. (Kushen). An orthogonal test L(9)(3)(4) including pressure, temperature, flow rate of CO(2) and the amount of modifier was performed to get the optimal conditions. The process was then scaled up by 30 times with a preparative SFE system under 25 MPa, 50 degrees C and a flow rate of CO(2) (2l/min) and the amount of modifier (0.04 ml/min). The crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of chloroform-methanol-2.3 x 10(-2)M NaH(2)PO(4) (27.5:20:12.5, v/v), and the collected fractions were analyzed by high-performance liquid chromatography (HPLC). Three kinds of quinolizidine alkaloids were obtained, yielding 10.02 mg of matrine, 22.07 mg of oxysophocarpine and 79.93 mg of oxymatrine with purities of 95.6, 95.8, 99.6% in one-step separation, respectively.

Detection and quantification of phnE gene from oil-contaminated soil samples by competitive quantitative PCR.

The phnE gene encoding catechol 2,3-dioxygenase belonging to the meta-cleavage pathway was selected as the marker gene and was detected and quantified from soil samples by competitive quantitative PCR. A PCR primer pair was designed based on the phnE gene to amplify the target DNA bands and competitor DNA bands. The phnE gene was detected in two samples of three. In samples S1 and S2, the phnE gene copy number was 6.2 x 10(7)/g soil and 5.8 x 10(7)/g soil, respectively. But no phnE gene was detected in sample S3. The target DNA bands were extracted and expressed. The results confirmed that the target DNA bands were the native phnE genes.

Synthesis and properties of cordycepin intercalates of Mg-Al-nitrate layered double hydroxides.

Layered double hydroxides (LDHs) were investigated as cordycepin delivery nanocarriers for the first time in this study. Negatively charged biomolecule-cordycepin was intercalated in the gallery spaces of [Mg-Al-NO(3)] as the charge-compensating species, which was confirmed by the results of XRD, FT-IR, TEM, CZE and electrophoretic mobility. Cell experiment suggested that the new bio-LDH nanohybrid could prevent cordycepin decomposition by adenosine deaminase. This new formulation could possibly be used as a novel form cordycepin intravenous injection.

[Study on the interaction of cordycepin and DNA].

The absorbance, fluorescence and Scatchard plots methods as well as the effect of phosphate group on the fluorescence intensity of the cordycepin-DNA-EB system were used to study the interaction of the antitumor compound cordycepin and DNA. It is obvious tiat there is hyperchromic effect and hypochromic effect with slight red shift on the subtracted UV spectrum. It proves that the adenine base of cordycepin can be inserted into the double-helix of DNA. The results of the fluorescence intensity that decreases after increases with a little blue shift on the fluorescence spectrum also proved this. At the same time the phosphate can affect the cordycepin-DNA-EB system. Finally that the result depended on the Scatchard equation indicates that cordycepin reacts electrostatically on phosphate backbone of DNA. There also exists an intercalation into the double-helix of DNA.

Conversion of an obligate autotrophic bacteria to heterotrophic growth: expression of a heterogeneous phosphofructokinase gene in the chemolithotroph Acidithiobacillus thiooxidans.

A plasmid pSDK-1 containing the Escherichia coli phosphofructokinase-1 gene (pfkA) was constructed, and transferred into Acidithiobacillus thiooxidans Tt-7 by conjugation. The pfkA gene from E. coli could be expressed in this obligately autotrophic bacterium but the enzyme activity (18 U g-1) was lower than that in E. coli (K12: 86 U g-1; DF1010 carrying plasmid pSDK-1: 97 U g-1). In the presence of glucose, the Tt-7 transconjugant consumed glucose leading to a better growth yield.

[A simple and effective method for extracting total RNA from domestic fungus].

With Flammulina velutipes material,an improved method was developed for extracting total RNA from domestic fungus that are rich in RNase,polyphenols, polymeric carbohydrates and proteoglycans. Phenol-chloroform-isoamyl alcohol were used twice to clear DNA and protein under higher concentration of denaturing solution and isopentanol, sodium acetate were used to precipitate RNA selectively. Pure and intact RNA can be effectively prepared by this method.

[Nucleoside from Cordyceps kyushuensis and the distribution of two active components in its different parts].

AIM: To rapidly separate and determine the nucleosides from natural and cultured Cordyceps kyushuensis Kob., and to compare the content of cordycepin and adenosine in different parts of Cordyceps kyushuensis Kob., which are the main nucleoside active components in medicinal fungus belonging to Cordyceps (Fr.) Link. METHODS: The nucleosides were separated and determined by the high performance capillary zone electrophoresis (CZE). Beckman P/ACE system MDQ apparatus equipped with a PDA detector and a uncoated fused-silica capillary (41 cm x 45 microns ID, 30 cm effective length) were used. The experimental conditions were as follows: the running buffer was borax solution (adjust to pH 9.4 with sodium hydroxide), applied voltage was 20 kV, operated temperature was 20 degrees C and the detector wavelength was 258 nm. The content of cordycepin and adenosine in the fruiting body, stroma and host worm of natural and cultured C. kyushuensis were respectively investigated and quantitatively analyzed. RESULTS: There are at least 8 kinds of nucleoside or nitrogen base in Cordyceps kyushuensis Kob. The content of cordycepin which is a bio-active substance with anti-tumor activity in C. kyushuensis is significantly higher than that in C. sinensis and C. militaris, and furthermore the cordycepin in the cultured C. kyushuensis is notably higher than the natural one. Adenosine was mainly found from the stroma of C. kyushuensis, While the cordycepin content is high in the stroma of both natural and cultured C. kyushuensis as well as in the host worm of the cultured one. CONCLUSION: There are some differences about the nucleoside components between the natural and cultured C. kyushuensis and between the different parts of them. With a high cordycepin content, C. kyushuensis should have a considerable medicinal potential.

Measurement of cordycepin and adenosine in stroma of Cordyceps sp. by capillary zone electrophoresis (CZE).

Capillary zone electrophoresis (CZE) was used to separate cordycepin and adenosine, and determine their concentration in stroma of Cordyceps sp. These two active components of natural and cultured Cordyceps kyushuensis were quantitatively analyzed and compared with those of cultured Cordyceps militaris. The results showed that the CZE method is a simple, rapid and sensitive method for the measurement of cordycepin and adenosine with good repeatability.