Main Nucleosides Research Articles

Schisandra chinensis bee pollen\u2019s chemical profiles and protective effect against H2O2-induced apoptosis in H9c2 cardiomyocytes

BackgroundSchisandra chinensis (Turcz.) Baill bee pollen extract (SCBPE) is often used as a functional food in China due to its good antioxidant property. However, its chemical compositions and effects on H9c2 cardiomyocytes against H2O2-induced cell injury still lacks of reports thus far. This study aimed to characterize the main components of SCBPE and investigate its protective effects against H2O2-induced H9c2 cardiomyocyte injury.MethodsThe main components of SCBPE were analyzed via ultraperformance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UPLC–QTOF MS/MS). The three main nucleosides in SCBPE were quantitatively analyzed via ultraperformance liquid chromatography–diode array detection. Furthermore, the potential mechanism by which SCBPE exerts protective effects against H2O2-induced H9c2 cardiomyocyte injury was explored for the first time via cell survival rate measurements; cell morphological observation; myocardial superoxide dismutase (SOD) activity and malondialdehyde (MDA) and glutathione (GSH) level determination; flow cytometry; and quantitative polymerase chain reaction.ResultsTwo carbohydrates, three nucleosides, and nine quinic acid nitrogen-containing derivatives in SCBPE were identified or tentatively characterized via UPLC–QTOF MS/MS. The nine quinic acid nitrogen-containing derivatives were first reported in bee pollen. The contents of uridine, guanosine, and adenosine were 2.4945 ± 0.0185, 0.1896 ± 0.0049, and 1.8418 ± 0.0157 μg/mg, respectively. Results of in vitro experiments showed that cell survival rate, myocardial SOD activity, and GSH level significantly increased and myocardial MDA level significantly decreased in SCBPE groups compared with those in H2O2 group. Cell morphology in SCBPE groups also markedly improved compared with that in H2O2 group. Results indicated that SCBPE protected H9c2 cardiomyocytes from H2O2-induced apoptosis by downregulating the mRNA expressions of Bax, cytochrome C, and caspase-3 and upregulating the Bcl-2 mRNA expression.ConclusionsThis study is the first to report that SCBPE could protect against oxidative stress injury and apoptosis in H2O2-injured H9c2 cells. Results indicated that the nucleosides and quinic acid nitrogen-containing derivatives could be the main substances that exert protective effects against H2O2-induced H9c2 cardiomyocyte injury.

Determination of the Main Nucleosides and Nucleobases in Natural and Cultured Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis, a recently described species of Ophiocordyceps that is associated with the larvae of Phassus nodus (Hepialidae) in the living root or trunk of the medicinal plant Clerodendrum cyrtophyllum, is the largest known Cordyceps species and is recognized as a desirable alternative for natural Ophiocordyceps sinensis. This study investigated the main nucleosides and nucleobases in natural and cultured Ophiocordyceps xuefengensis. The contents of the nucleosides and nucleobases in the natural and cultured samples were determined by reverse phase HPLC. The highest concentration of adenosine was found in the natural fruit body and the cultured stroma, with almost no adenosine in the cadaver of Phassus nodus. The contents of adenine, guanosine, uridine and uracil in the cultured mycelium were significantly higher than those in the natural sample. Inosine was only detected in the natural samples. Thymidine and 2-deoxyadenosine were only found in the cadaver of Phassus nodus. Cordycepin was not detected in the five samples examined. These results suggested that the cultured mycelium and cultured stroma of Ophiocordyceps xuefengensis might be a promising substitute for natural O. xuefengensis.

Quantitative analysis of nucleosides in four Cordyceps genus by HPLC

To compare the main nucleosides in Cordyceps genus herbs (C. sinensis, C. millitaris, Hirsutella sinensis and C. sobolifera), an HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine in Cordyceps genus herbs was developed. The sample was extracted with 0.5% phosphoric acid solution to prepare test solution. The separation was performed on a Zorbax SB-Aq (4.6 mm×150 mm, 5 μm) column with gradient elution by 0.04 mol•L⁻¹ potassium dihydrogen phosphate solution and acetonitrile, column temperature 30 ℃,flow rate 0.8 mL•min⁻¹,and detection wavelength 260 nm. The content of nucleosides in four Cordyceps genus herbs was evaluated by fingerprint analysis and hierarchical cluster analysis (HCA). The calibration curves of five nucleosides showed good linear regression (r>0.99) and the average recoveries were between 95.0% and 105.0%. The contents of the five nucleosides in the four Cordyceps genus herbs were different and could be obviously distinguished by HCA. The fingerprint analysis result showed that the similarity between C. sinensis and the others was less than 0.9. The method was accurate and reliable, which can be used for quality control of Cordyceps genus herbs.

Extreme nucleoside concentrations in a marine annelid: A novel nucleoside storing cell in the polychaete Nereis virens

A new type of free coelomic cell was isolated from the coelomic fluid of the polychaete Nereis virens. This cell type was present only during sexual maturation and only in males with fully developed spermatozoa. In vivo acridine orange staining and electron microscopy showed that this cell type consists mainly of a large acidic vacuole with a very low proportion of cytoplasm. This cell type stores extremely high concentration of purine nucleosides reaching concentrations of >700 millimolar on a cell weight basis. The nucleoside concentrations are the highest reported from any living cell so far. Inosine (280-711 micromol/g cell mass), guanosine (8-109 micromol/g), deoxyinosine (3-26 micromol/g) and deoxyguanosine (0.05-0.70 micromol/g) were the main nucleosides. The spectrum of nucleosides reflected that of the surrounding coelomic fluid suggesting their exogenous origin. In culture, labeled guanosine was effectively concentrated by the cells. The vacuole is the most likely storage compartment for the nucleosides and its acidic pH provides higher nucleoside solubility and prevents back diffusion by protonation of the nucleosides.

Analysis of the Main Nucleosides in Cordyceps Sinensis by LC/ESI-MS

A sensitive, selective and reliable liquid chromatography-mass spectrometry coupled with electrospray ionization interface method for simultaneous separation and determination of thymine, adenine, adenosine and cordycepin in Cordyceps sinensis has been established. The optimum separation for these analytes was achieved using a gradient elution system and a 2.0 × 150 mm Shimadzu VP-ODS column. 2-Chloroadenosine was used as internal standard for this assay. [M+H]+ions at m/z 127, 136, 268, 252 and 302 were chosen and selective ion monitoring (SIM) mode was used for quantitative analysis of the four main nucleosides. The regression equations were linear in the range of 1.0–117.5 μg·mL-1 for thymine, 1.8-127.0 μg·mL-1 for adenine, 0.6-114.0 μg·mL-1 for adenosine and 0.5-107.5 μg·mL-1 for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were 1.0 and 0.2 μg·mL-1 for thymine, 1.8 and 0.6 μg·mL-1 for adenine, 0.6 and 0.1 μg·mL‑1 for adenosine and 0.5 and 0.1 μg·mL-1 for cordycepin, respectively. The recoveries of the four nucleosides ranged from 98.47 to 99.32%. The developed method was successfully used to determine nucleosides in Cordyceps sinensis from different sources.

Simultaneous determination of six main nucleosides and bases in natural and cultured Cordyceps by capillary electrophoresis

A simple method is described for simultaneous determination of six main nucleosides and bases including adenine, uracil, adenosine, guanosine, uridine and inosine in Cordyceps by capillary electrophoresis (CE). Chemometric optimization based on central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: buffer concentration, pH and concentration of acetonitrile as organic modifier. Resolution ( R S) was employed to evaluate the response function. A running buffer composed of 500 mM boric acid, adjusted pH to 8.6 with sodium hydroxide and 12.2% acetonitrile as modifier was found to be the most appropriate for the separation. The contents of the six components were determined by using adenosine monophosphate as an internal standard. Furthermore, hierarchical clustering analysis based on characteristics of 32 peaks in CE profiles from the tested 12 samples showed that natural and cultured Cordyceps were in different clusters. Adenosine and inosine were extracted as markers for discrimination of natural Cordyceps. The result of clustering based on the two peaks characteristics was in excellent agreement with that based on 32 peaks’. Thus, adenosine and inosine could be used as markers for quality control of natural and cultured Cordyceps.

Nucleoside uptake in male germ cells of the polychaete Nereis virens

Summary Uptake of inosine and guanosine was measured in male germ ceils of the polychaete Nereis virens at different stages of development. In spermatogonia I (spg I) and spermatid stages, total inosine uptake at 12°C and ambient concentrations of 100 μmol/1 was relatively low (10–50 nmol·ml of packed cell volume pcv−l·h−1). A rapid increase (150–300 nmol·ml of pcv-1·h−1) was found during transition from spg I to spg II with a subsequent decline to low values (10–30 nmol·ml of pcv−1·h−1) in spermatocyte and spermatid stages. This transient increase may be related to the proliferative activity of spg I stages leading to spg II stages, which increases the demand of purine precursors for nucleic acid synthesis. Inosine uptake of spermatozoa stages was extremely variable, and high uptake rates (10–300 nmol·ml of pcv−1·h−1) were found in some, but not in all, cases. In all stages the uptake rates for guanosine remained at a lower level (below 75 nmol·ml of pcv−1·h−1), and no clear stage specific pattern was evident except for low values (<10 nmol·ml of pcv −1·h−1) in motile spermatozoa. For both nucleosides, two uptake components were identified: a linear component (measured at medium concentrations up to 300 μmol/l) and a saturable component (app. km: 9–60 μmol/l). The latter was inhibited by iodoacetamide suggesting the presence of an ATP-dependent facilitated transport mechanism. The presence of the two uptake components was related to the stage of gamete development: the saturable uptake component present in early spg I changed to a linear uptake in late spg I. In spg II stages, both uptake components were present while in spermatocytes, only the saturable component was present. Spermatids and early spermatozoa showed only a linear uptake. In the coelomic fluid, guanosine (0.5–20 μmol/l) and inosine (>3 μmol/l) were the main nucleosides; during spg II and spermatocyte stages, inosine reached high concentrations (100–1000 μmol/l).

The Separation of the Major Ribonucleotides, Nucleosides, and Bases in Reverse-Phase Ion-Pair Chromatography

Abstract The separation of 12 major nucleotides was achieved on a reversed-phase HPLC system with the radially compressed NovaPak C18 column and tetrabutylammonium phosphate (TBA) ion pairing reagent by acetonitrile gradient elution. The effect of tetrabutylammonium phosphate and acetonitrile on the retention of the major nucleotides in this system was studied. It was found that in k' decreases in a proportion to the concentration of acetonitrile in the mobile phase. The maximum of nucleotide retention was found to be at TBA concentration of about 2 mM, the amount of acetonitrile in the mobile phase being constant. A simultaneous separation of main nucleotides, nucleosides, and bases was achieved under optimum conditions.

Pyrimidine nucleoside transformations via anhydronucleosides.

Abstract