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

Ophiocordyceps xuefengensis, a recently described species of Ophiocordycepsthat is associated with the larvae of Phassusnodus (Hepialidae) in the living root or trunk of the medicinal plant Clerodendrumcyrtophyllum, isthe largest known Cordycepsspecies and is recognized as a desirable alternative for natural Ophiocordycepssinensis. This study investigated the main nucleosides and nucleobases in natural and cultured Ophiocordycepsxuefengensis. 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 Phassusnodus. 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 Phassusnodus. Cordycepin was not detected in the five samples examined. These results suggested that the cultured mycelium and cultured stroma of Ophiocordycepsxuefengensis might be a promising substitute for natural O. xuefengensis.

Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines.

Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.

Artificial receptors for the extraction of nucleoside metabolite 7-methylguanosine from aqueous media made by molecular imprinting.

A series of imprinted polymers targeting nucleoside metabolites, prepared using a template analogue approach, are presented. These were prepared following selection of the optimum functional monomer by solution association studies using (1)H NMR titrations whereby methacrylic acid was shown to be the strongest receptor with and affinity constant of 621±51Lmol(-1)vs. 110±16Lmol(-1) for acrylamide. The best performing polymers were prepared using methanol as porogenic co-solvent and although average binding site affinities were marginally reduced, 2.3×10(4)Lmol(-1)vs. 2.7×10(4)Lmol(-1) measured for a polymer prepared in acetonitrile, these polymers contained the highest number of binding sites, 5.27µmolg(-1)vs. 1.64µmolg(-1), while they also exhibited enhanced selectivity for methylated guanosine derivatives. When applied as sorbents in the extraction of nucleoside derivative cancer biomarkers from synthetic urine samples, significant sample clean-up and recoveries of up to 90% for 7-methylguanosine were achieved.

Hydrophilic organic/salt-containing aqueous two-phase solvent system for counter-current chromatography: a novel technique for separation of polar compounds.

Hydrophilic organic/salt-containing aqueous two-phase system composing of ethanol, water and ammonium sulfate for separation polar compounds was investigated on multilayer coil associated with J-type HSCCC devices. Compared to the classical polar solvent system based on 1-butanol-water or PEG1000-ammonium sulfate-water, the water content of upper phase in ethanol-ammonium sulfate-water systems was from 53.7% to 32.8% (wt%), closed to PEG1000-ammonium sulfate-water aqueous two-phase systems and higher than 1-butanol-water (22.0%, wt%). Therefore, the polarity of ethanol-ammonium sulfate-water is in the middle of 1-butanol-water and PEG-ammonium sulfate-water system, which is quite good for separating polar compounds like phenols, nucleosides and amino acids with low partition coefficient in 1-octanol-water system. The retention of stationary phase in four elution mode on type-J counter-current chromatography devices with multilayer coil column changed from 26% to 71%. Hydrodynamic trend possess both intermediate and hydrophilic solvent system property, which closely related to the composition of solvent system. The applicability of this system was demonstrated by successful separation of adenosine, uridine guanosine and cytidine.

Identification and analysis of the constituents in an aqueous extract of Tricholoma matsutake by HPLC coupled with diode array detection/electrospray ionization mass spectrometry.

The main constituents in an aqueous extract of Tricholoma matsutake (Tm) were identified by high-performance liquid chromatography coupled with diode array detection and electrospray ionization time-of-flight mass spectrometry (HPLC-DAD/TOF-MS) and ion trap mass spectrometry (HPLC-DAD/Trap-MSn). The main factors in the extraction process which affect the yields of nutrients were optimized by single-factor experiments and orthogonal experiment design. In total, 12 constituents were identified from the aqueous extract of Tm, including tyrosine, cytidine, uridine, eritadenine, phenylalanine, nicotinamide, inosine, guanosine, tryptophan, adenosine, 5'-deoxy-5'-methylthioadenosine and riboflavin. The optimized extraction conditions were: the ratio of water to sample was 10 : 1 (v/w), Tm was extracted by ultrasonic-assisted extraction for 10 min, followed by water bath heating at 60 °C for 1 h. Among these extraction factors, the heating temperature is significant based on analysis of variance (ANOVA). The yields of nutrients were affected dramatically at high temperature leading to the loss of nutrients, especially for nucleosides and some amino acids.

Study of retention behaviour and mass spectrometry compatibility in zwitterionic hydrophilic interaction chromatography for the separation of modified nucleosides and nucleobases.

A study has been made of the chromatographic behaviour of modified nucleosides and nucleobases using different stationary phases with functional groups of polar nature, all of them compatible with aquoorganic mobile phases. The stationary phases assayed were a pentafluorophenylpropyl (PFP) column for reverse phase separation, and another two for hydrophilic interaction chromatography (HILIC) separation. Six modified nucleosides and nucleobases (hydroxylated and methylated derivatives) were chosen as the target analytes. In the study, chromatographic resolution as well as the sensitivity in detection by mass spectrometry were taken into account. The results obtained showed that the zwitterionic (ZIC-HILIC) column was the most suitable one for the separation of these analytes. From the study of the different parameters affecting separation it may be concluded that in the ZIC-HILIC column separation is based on a mechanism of partition and interaction through weak electrostatic forces.

Isolation of adenosine, iso-sinensetin and dimethylguanosine with antioxidant and HIV-1 protease inhibiting activities from fruiting bodies of Cordyceps militaris.

According to previous studies, a close relationship between oxidative stress and AIDS suggests that antioxidants might play an important role in the treatment of AIDS. Cordyceps militaris was selected from nine edible mushrooms by assay of inhibition of erythrocyte hemolysis. Macroporous adsorption resin and HPLC were used to purify three micromolecular compounds named L3a, L3b and L3c. L3a was identified to be adenosine with the molecular formula C(10)H(13)N(5)O(4); L3b was 6,7,2',4',5'-pentamethoxyflavone with the molecular formula C(20)H(20)O(7), and L3c was dimethylguanosine with the molecular formula C(12)H(17)N(5)O(5). The compound 6,7,2',4',5'-pentamethoxyflavone was first isolated from C. militaris. The assay of inhibition of HIV-1 protease (HIV-1 PR) was based on the fact that the expression of this enzyme can inhibit the growth of E. coli. This is a new screening system for HIV-1 PR inhibitors. Both L3a and L3b showed high inhibition to HIV-1 PR. These compounds could be new anti-HIV-1 PR drugs.

[Determination of isoguanosine in Semen Crotonis Tiglii by high-performance liquid chromatography].

OBJECTIVE: To develop a high-performance liquid chromatographic method for the determination of isoguanosine in Semen Crotonis Tiglii. METHODS: The determination was done by using reverse-phase high-performance liquid chromatography with a Hedera ODS-2 column (4.6 mm x 250 mm, 5 microm). Elution was employed with the mobile phase of acetonitrile-methanol-water (1:4:95) at flow rate of 1.0 mL/min. The detection wavelength was set at 292 nm and the column temperature was 25 degrees centigrade. Injection volume was 10 microL. RESULTS: The linear range of isoguanosine was from 0.161 to 0.967 mg. The correlation coefficient of the calibration curves was 0.999 8. The average recovery rate was 98.78% with relative standard deviation of 0.02%. CONCLUSION: The results show that the method is simple, accurate, and repeatable, and it is suitable for quality control of Semen Crotonis Tiglii.

Effects of sample preparation methods on the quantification of nucleosides in natural and cultured Cordyceps.

Sample preparation is the first and very important step, which can greatly influence the repeatability and accuracy of the analysis. To date, several sample preparation methods with different solvents have been used for quantitative determination of nucleosides in Cordyceps, but their data are greatly various. In this study, five nucleosides, including adenosine, guanosine, inosine, uridine and cordycepin, in Cordyceps were determined using three extraction methods i.e. organic solvent pressurized liquid extraction, boiling water extraction and ambient temperature water extraction and high performance liquid chromatography (HPLC)-diode array detection (DAD). The similar results were obtained when organic solvent pressurized liquid extraction and boiling water extraction were applied. However, the amounts of nucleosides in natural C. sinensis and cultured C. militaris extracted with ambient temperature water were greatly increased except those of adenosine in natural C. sinensis and cordycepin in cultured C. militaris. In addition, the amount of investigated nucleosides in cultured C. sinensis had no obvious variation among the three extraction methods. The results suggest that sample preparation has significant effect on the quantification of nucleosides in Cordyceps.

[Chemical study of nucleosides on Cordyceps militaris].

OBJECTIVE: To investigate the nucleosides of Cordyceps militaris. METHODS: The primary extract technique of nucleosides from Cordyceps militaris was founded, the nucleosides were isolated by chromatography. RESULTS: Four compounds were isolated from the mycelium of Cordyceps militaris. They were identified as uridine (1), adenosine (2), guanosine (3) and p-tyrosine (4), respectively. CONCLUSION: Compounds 1, 2 and 3 are the main nucleosides in Cordyceps militaris.

On-line hyphenation of quantum cascade laser and capillary electrophoresis.

We report the first successful hyphenation of a Fabry Pérot quantum cascade (QC) laser to a capillary electrophoresis system. This involved use of a dedicated IR-transparent flow cell, made of CaF2, constructed by means of SU-8 based lithography and low temperature wafer bonding techniques. Adenosine, guanosine, xanthosine and adenosine-5'-monophosphate were separated in a borate-containing separation electrolyte (10 mM, pH 9.3). Functional group (carbohydrate) detection was accomplished by use of the 1080 cm(-1) emission line of the available QC-laser. The assessable optical path length could be increased, from the normally available 10-15 microm in CE-FTIR analyses, to 60 microm using this powerful mid-infrared laser and aqueous solutions.

[Identification of 5 constituents of the aqueous extract of Isatis indigotica by HPLC-MS2].

OBJECTIVE: To identify five constituents in the aqueous extract of Isatis indigotica Fort. METHODS: After separation of the aqueous extract of Isatis indigotica Fort. by HPLC, the eluates of five peaks were collected separatively and analysed by MS2. UV spectra and MS2 were compared with those of reference standards of cytidine, uridine, guanosine xanthine and hypoxanthine. RESULTS: Each HPLC elute of the aqueous extract had same retention time, UV spectra and fragment pattern in the MS2 spectrum as the corresponding standards. CONCLUSION: Five constituents of the aqueous extract of Isatis indigotica Fort. are identified as cytidine, hypoxanthine, uridine, xanthine and guanosine.

[Study on the chemical constituents of Selaginella tamariscina (Beauv.) Spring].

AIM: To study the chemical constituents of Selaginella tamariscina (Beauv.) Spring. METHODS: Various chromatographic techniques were used to separate and purify the chemical constituents. Their physico-chemical properties and spectral data were used to elucidate the structures. RESULTS: Four compounds were isolated from the n-BuOH fraction of the water-extracts. Their structures were identified as 1-hydroxy-2-[2-hydroxy-3-methoxy-5-(1-hydroxyethyl)-phenyl]-3-(4-hydroxy-3,5-dimethoxy)-propane-1-O-beta-D-glucopyranoside (tamariscinoside B, I), adenosine (II), guanosine (III), arbutin (IV). CONCLUSION: Tamariscinoside B (I) is a new compound, while the others were isolated from Selaginella for the first time.

Structure of wyosine, the condensed tricyclic nucleoside of torula yeast phenylalanine transfer ribonucleic acid.

Large-scale isolation of the minor nucleoside wyosine of torula yeast tRNA(Phe) was accomplished by a combination of enzymatic digestion and reversed-phase chromatography: the wyosine-containing nucleotide fraction, which was obtained by partial digestion of unfractionated tRNA (1 g) with nuclease P1, was concentrated by reversed-phase column chromatography followed by complete digestion with nuclease P1/alkaline phosphatase. The nucleoside mixture thus obtained was purified by reversed-phase HPLC, providing wyosine (70 microg). Comparison of this nucleoside with a chemically synthesized authentic sample has unambiguously established that the structure of wyosine is 4,6-dimethyl-3-beta-D-ribofuranosyl-3,4-dihydro-9H-imidazo[1,2-a]purin-9-one (2).

Separation of inosine, hypoxanthine, and guanosine by high-performance liquid chromatography on silica.

The separation of inosine (Ino), hypoxanthine (Hyp), and guanosine (Guo) on silica has been studied. In adsorption normal-phase systems the peak shapes are unsatisfactory; a low selectivity has been observed for the Ino-Hyp and Guo-Hyp pairs. Chromatograms can be significantly improved if systems with a mixed partition-adsorption retention mechanism are applied.

Simultaneous immunoaffinity purification of O6-methyl, O6-ethyl-, O6-propyl- and O6-butylguanine and their analysis by gas chromatography/mass spectrometry.

A sensitive and specific method has been developed for the simultaneous analysis of different O6-alkylguanines. The cross-reactivity of two different antibodies raised against O6-methylguanosine and O6-butylguanosine for a series of O6-alkylguanines was exploited for the immunoaffinity purification of biological samples before quantitative analysis by gas chromatography/mass spectrometry. The method can be applied to the detection of O6-alkylguanines in DNA and appears to be useful for studying chemical carcinogen mechanisms in animals and possibly for the detection of human exposure to alkylating agents.

Isolation of isoguanosine from Croton tiglium and its antitumor activity.

This paper describes the isolation of isoguanosine from Croton tiglium L. and its cytotoxic effect against several tumor cell lines in culture and newly reports that isoguanosine has an antitumor activity against implanted S-180 ascitic tumor mice. Isoguanosine is effective at the dose of 24 mg/kg/day x 5, with T/C value of 168%. Isoguanosine inhibits the growth of S-180 and Ehrlich solid tumor in mice at the optimal doses of 96 mg/kg/day x 12 and 48 mg/kg/day x 12, with 1-T/C values of 65% and 60%, respectively.

Quantification of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection.

7-Methylguanine (7-meG) could be a useful marker of recent past exposure to environmental methylating agents for use in epidemiological studies. A method is described that is appropriate for such an application. 7-meG was released from DNA by thermal hydrolysis under conditions (pH 9, 70 degrees C, 8 h) that preferentially released the base from DNA rather than RNA and, following immunopurification using antibodies specific for this DNA adduct, quantification was achieved either by HPLC with electrochemical detection (ECD) or by ELISA. The detection limits of the two approaches were 0.5 and 2 pmol 7-meG/DNA sample respectively. 7-meG was analysed in DNA samples contaminated with known amounts of RNA to test the possible interference in the analysis by the minor modified nucleoside 7-methylguanine, which is present as a normal component of RNA. 7-meG levels measured in human pancreas and untreated rat liver DNA were between 2 and 7 pmol 7-meG/mumol guanosine and this level could not be explained by RNA contamination. The combination of immunoaffinity purification and HPLC with ECD provides a method that is sensitive and specific for 7-meG and suitable for integration into molecular epidemiological studies.

Structure of the archaeal transfer RNA nucleoside G*-15 (2-amino-4,7-dihydro- 4-oxo-7-beta-D-ribofuranosyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboximi dam ide (archaeosine)).

A number of post-transcriptional modifications in tRNA are phylogenetically characteristic of the bacterial, eukaryal, or archaeal domains, both with respect to sequence location and molecular structure at the nucleoside level. One of the most distinct such modifications is nucleoside G*, located in archaeal tRNA at position 15, which in bacterial and eukaryal tRNAs is a conserved site involved in maintenance of the dihydrouridine loop-T-loop tertiary interactions. G* occurs widely in nearly every branch of the archaeal phylogenetic domain, in contrast to its absence in all reported bacterial and eukaryal tRNA sequences. The structure of G*-15 is 2-amino-4,7-dihydro-4-oxo-7-beta-D-ribofuranosyl-1H- pyrrolo[2,3-d]pyrimidine-5-carboximidamide (7-formamidino-7-deazaguanosine), which is a non-purine, non-pyrimidine ribonucleoside; its structure thus reflects extensive modification beyond the guanine-15 specified by corresponding gene sequences. The structure was established by mass spectrometry, and in particular from collision-induced dissociation mass spectra of derivatives formed by microscale permethylation, and is confirmed by chemical synthesis.

Direct assay method for guanosine 5'-monophosphate reductase activity.

A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).