Comparative Analysis of Carbohydrates, Nucleosides and Amino Acids in Different Parts of Trichosanthes kirilowii Maxim. by (Ultra) High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry and Evaporative Light Scattering Detector Methods.

Trichosanthes kirilowii Maxim. is one of the original plants for traditional Chinese medicines Trichosanthis Fructus, Trichosanthis Semen, Trichosanthis Pericarpium and Trichosanthis Radix. Amino acids, nucleosides and carbohydrates are usually considered to have nutritional value and health-care efficacy. In this study, methods involving high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD), UV-visible spectrophotometry and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) were established for quantifying carbohydrates (fructose, glucose, stachyose, raffinose and polysaccharide), fourteen nucleosides and twenty one amino acids. Moreover, sixty-three samples from nine different parts, including pericarp, seed, fruit pulp, stem, leaf, main root, main root bark, lateral root and lateral root bark of T. kirilowii from different cultivated varieties were examined. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. The results showed that the average content of total amino acids in roots (15.39 mg/g) and root barks (16.38 mg/g) were relatively higher than for others. Contents of nucleosides in all parts of T. kirilowii were below 1.5 mg/g. For carbohydrates, fruit pulp has a higher content than others for glucose (22.91%), fructose (20.63%) and polysaccharides (27.29%). By using partial least-squared discriminate analysis (PLS-DA), Variable importance in the projection (VIP) plots and analysis of variance (ANOVA) analysis, the characteristic components of the different organs (fruit, stems and leaves, roots) were found. This analysis suggested there were potential medicinal and nutritive health care values in various parts of the T. kirilowii, which provided valuable information for the development and utilization of T. kirilowii.

Nucleoside and N-acetyldopamine derivatives from the insect Aspongopus chinensis.

Two new nucleoside derivatives, named asponguanosines A and B (1 and 2), three new N-acetyldopamine analogues, aspongamides C-E (3-5), one new sesquiterpene, aspongnoid D (6), and three known compounds were isolated from the medicinal insect Aspongopus chinensis. Their structures including absolute configurations were assigned by using spectroscopic methods and ECD and 13C NMR calculations. Biological activities of compounds 3-7 towards human cancer cells, COX-2, ROCK1, and JAK3 were evaluated.

Preparation and evaluation of a reversed-phase/hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase functionalized with dopamine-based dendrimers.

In this work, a novel dendritic stationary phase was synthesized by the repeated grafting of 1,4-butanediol diglycidyl ether (BDDE) and dopamine (DA) on the surface of silica for performing mixed-mode high-performance liquid chromatography (MHPLC). Elemental analysis (EA), thermogravimetric analysis (TGA) and Fourier transform infrared spectrometry (FT-IR) showed the successful preparation of the dendritic stationary phase. The prepared stationary phase showed the retention mechanisms of reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and ion-exchange chromatography (IEC) under different mobile phase conditions. In detail, alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs) and hydrophobic positional isomers were separated successfully in the RPLC mode. The baseline separation of nucleobases, nucleosides and flavonoids was achieved under HILIC mode, respectively. Meanwhile, some acidic and basic analytes were used to evaluate the IEC mode. The effects of different chromatographic conditions, such as acetonitrile content, salt concentration and pH in the mobile phase, on the different chromatographic modes were also investigated. In addition, the application of the mixed-mode dendritic stationary phase was demonstrated by the analysis of traditional Chinese medicine (TCM), including Carthamus tinctorius L. and Abelmoschus manihot (Linn.) Medicus. Interestingly, the stationary phase also has the ability for the capture and separation of boric acids. These meaningful applications confirmed that the mixed-mode dendritic stationary phase can be potentially applied in the analysis of complex samples.

Quality Evaluation of Apocyni Veneti Folium from Different Habitats and Commercial Herbs Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Apocyni Veneti Folium (AVF) is a kind of staple traditional Chinese medicine with vast clinical consumption because of its positive effects. However, due to the habitats and adulterants, its quality is uneven. To control the quality of this medicinal herb, in this study, the quality of AVF was evaluated based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis. A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was developed for the simultaneous determination of a total of 43 constituents, including 15 flavonoids, 6 organic acids, 13 amino acids, and 9 nucleosides in 41 Luobumaye samples from different habitats and commercial herbs. Furthermore, according to the contents of these 43 constituents, principal component analysis (PCA) was employed to classify and distinguish between AVF and its adulterants, leaves of Poacynum hendersonii (PHF), and gray relational analysis (GRA) was performed to evaluate the quality of the samples. The proposed method was successfully applied to the comprehensive quality evaluation of AVF, and all results demonstrated that the quality of AVF was higher than the PHF. This study will provide comprehensive information necessary for the quality control of AVF.

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.

Quality Evaluation of Pseudostellariae Radix Based on Simultaneous Determination of Multiple Bioactive Components Combined with Grey Relational Analysis.

Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine (TCM) with vast clinical consumption because of its positive effects. However, little attention has been devoted to simultaneous analysis of its bioactive components for quality control of PR based on its different harvesting times, different growing habitats, and different processing methods. In this research, the quality of PR was evaluated based on simultaneous determination of multiple bioactive components combined with grey relational analysis (GRA). A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established to simultaneously determine the contents of 30 components in PR, including two cyclopeptides, 12 nucleosides, and 16 amino acids. Furthermore, grey relational analysis was performed to evaluate the quality of PR samples according to the contents of these 30 components. The results showed that the quality of PR harvested in 6 August 2013, cultivated in Jurong, Jiangsu, and treated by oven drying 60 °C was better than that of other PR samples. The proposed method is useful for the overall assessment on the quality of PR, and this study provides valuable information for revealing the dynamic change laws of metabolite accumulation in PR and choosing the most suitable harvesting time and reasonable processing method of PR to obtain the best quality.

Simultaneous Determination of 16 Nucleosides and Nucleobases in Euryale ferox Salisb. by Liquid Chromatography Coupled with Electro Spray Ionization Tandem Triple Quadrupole Mass Spectrometry (HPLC-ESI-TQ-MS/MS) in Multiple Reaction Monitoring (MRM) Mode.

In this study, a simple, rapid, efficient analytical method was established for the qualification and quantification of 16 nucleosides and nucleobases in Euryale ferox Salisb. by using liquid chromatography coupled with electrospray ionization tandem triple quadrupole mass spectrometry (HPLC-ESI-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Ideal separation of 16 target compounds was achieved on Xbridge Amide HILIC column (4.6 × 150 mm, 3.5 µm) with gradient elution in 11 min by optimized conditions. Variations of nucleosides and nucleobase in samples from different cultivation regions ranging from 190.50 to 1594.30 µg/g were obvious. The total nucleoside contents were higher than total nucleobases, especially in the compositions of guanosine, cytidine and 2'-deoxyguanosine. Samples 1-18 with dense thorns were better characters than samples 19-26 without thorns in terms of nucleosides and nucleobases concentrations in general. The limits of detection (LODs) and quantification (LOQs) for 16 analytical substances were investigated to be 0.11-6.33 ng/mL and 0.35-21.1 ng/mL, respectively. And the method was first applied to large aquatic plants with good linearity, precision, repeatability and accuracy. All present information provided a scientific and rational reference for quality assessment and control of Euryale ferox Salisb.

Multivariate analysis of chromatographic retention data as a supplementary means for grouping structurally related compounds.

In the present study a series of 45 metabolite standards belonging to four chemically similar metabolite classes (sugars, amino acids, nucleosides and nucleobases, and amines) was subjected to LC analysis on three HILIC columns under 21 different gradient conditions with the aim to explore whether the retention properties of these analytes are determined from the chemical group they belong. Two multivariate techniques, principal component analysis (PCA) and discriminant analysis (DA), were used for statistical evaluation of the chromatographic data and extraction similarities between chemically related compounds. The total variance explained by the first two principal components of PCA was found to be about 98%, whereas both statistical analyses indicated that all analytes are successfully grouped in four clusters of chemical structure based on the retention obtained in four or at least three chromatographic runs, which, however should be performed on two different HILIC columns. Moreover, leave-one-out cross-validation of the above retention data set showed that the chemical group in which an analyte belongs can be 95.6% correctly predicted when the analyte is subjected to LC analysis under the same four or three experimental conditions as the all set of analytes was run beforehand. That, in turn, may assist with disambiguation of analyte identification in complex biological extracts.

Identification of nucleosides and nucleobases from cultured Cordyceps militaris.

Four nucleosides and seven nucleobases were isolated from the BuOH subfraction of the extract of cultured Cordyceps militaris; one of them, 6-acetylpurine (1) is a new natural compound. The structure of I was determined on the basis of HR-ESI-MS, and I D and 2D NMR spectroscopic analysis.

Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes.

BACKGROUND: Nikkomycins are competitive inhibitors of chitin synthase and inhibit the growth of filamentous fungi, insects, acarids and yeasts. The gene cluster responsible for biosynthesis of nikkomycins has been cloned and the biosynthetic pathway was elucidated at the genetic, enzymatic and regulatory levels. RESULTS: Streptomyces ansochromogenes ΔsanL was constructed by homologous recombination and the mutant strain was fed with benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid. Two novel nikkomycin analogues were produced when cultures were supplemented with nicotinic acid. These two compounds were identified as nikkomycin Px and Pz by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Bioassays against Candida albicans and Alternaria longipes showed that nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by Streptomyces ansochromogenes 7100 (wild-type strain). Moreover, nikkomycin Px and Pz were found to be more stable than nikkomycin X and Z at different pH and temperature conditions. CONCLUSIONS: Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with the sanL inactivated mutant of Streptomyces ansochromogenes 7100. Although antifungal activities of these two compounds are similar to those of nikkomycin X and Z, their stabilities are much better than nikkomycin X and Z under different pHs and temperatures.

Characterization of nucleobases and nucleosides in the fruit of Alpinia oxyphylla collected from different cultivation regions.

The fruit of Alpinia oxyphylla, known as Yizhi, Yakuchi and Ikji in Chinese, Japanese, and Korean, respectively, has been utilized as an important drug for the treatment of diarrhea, dyspepsia, spermatorrhea, kidney asthenia and abdominal pain in East Asian traditional medicine for thousands of years. Since the therapeutic effects of A. oxyphylla are attributed to multiple components and nucleobases and nucleosides exhibit various bioactivities, it is necessary to explore the chemical characterization of nucleobases and nucleosides in this herb. Herein, 10 common nucleobases and nucleosides, including cytidine, adenosine, thymidine, inosine, guanosine, 2'-deoxyinosine, guanine, adenine, cytosine, and hypoxanthine, were quantified simultaneously in the fruit of A. oxyphylla collected from different geographical regions. Changes in their contents were discussed, and hierarchical cluster analysis (HCA) was performed to classify all samples on the basis of the contents of the investigated analytes. The results indicated that there was a large variation in the contents of nucleobases and nucleosides among the herbs from different regions, and the samples collected from the same cultivation region were mostly classified in one cluster. The method can be used for comprehensive quality evaluation of A. oxyphylla.

Simultaneous determination of nucleosides and their bases in Cordyceps sinensis and its substitutes by matrix solid-phase dispersion extraction and HPLC.

Nine nucleosides and nucleobases, including uracil, adenine, thymine, uridine, adenosine, thymidine, cytidine, guanosine, and cordycepin in natural Cordyceps sinensis, cultured Cordyceps mycelia, and Cordyceps fruiting bodies were extracted by matrix solid-phase dispersion (MSPD) and determined by HPLC. The experimental conditions for the MSPD extraction were optimized. Florisil was used as dispersant, petroleum ether as washing solvent, and methanol as elution solvent. The Florisil-to-sample ratio was selected to be 4:1 and no additional clean-up sorbent was needed. The calibration curves had good linear relationships (r > 0.9997). The LOD and LOQ were in the range of 12～79 and 41～265 ng/mL, respectively. The intra- and interday precision were lower than 8.3%. The recoveries were between 61.5 and 93.2%. The present method consumed less sample compared with ultrasonic extraction and heating reflux extraction (HRE). The extraction yields obtained by using the present method are much higher than those obtained by UE and comparable to those obtained by HRE.

Characterisation of nucleosides and nucleobases in Mactra veneriformis by high performance liquid chromatography coupled with diode array detector-mass spectrometry (HPLC-DAD-MS).

Mactra veneriformis has been used as sea food and traditional Chinese medicine (TCM) for thousands of years in China. In the present study, a high performance liquid chromatograph coupled with photodiode array detector and electrospray ionisation-mass spectrometer (HPLC-DAD-ESI-MS) method was established for detection of the nucleosides and nucleobases in M. veneriformis from four aquaticultural area of Jiangsu during different harvest time of one year. The validated method was successfully applied to identifying 10 nucleosides and nucleobases in 48 M. veneriformis samples. Quantitative analysis showed that nucleosides and nucleobases are rich in all M. veneriformis samples. However, their contents vary in different areas and harvest times. Principal component analysis (PCA) was used to classify the 48 samples based on the contents of the nucleosides and nucleobases. As a result, the samples could be mainly clustered into four groups, which was similar as aquaticultural areas classification. Based on the results, present method might be applicable for the quality control of M. veneriformis, or even other marine shellfish aquiculture and their products, and the quality of M. veneriformis might be more related with aquaticultural areas.

Determination of nucleosides and nucleobases in different species of Cordyceps by capillary electrophoresis-mass spectrometry.

In present study, a capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine, guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2'-deoxyuridine, uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside as an internal standard (IS). The CE separation conditions and MS parameters were optimized systematically for achieving good CE resolution and MS response of the investigated compounds. The optimum CE electrolyte was 100 mM formic acid containing 10% (v/v) methanol. The optimum MS parameters were as follows: 75% (v/v) methanol containing 0.3% formic acid with a flow rate of 3 microL/min was selected as the sheath liquid; the flow rate and temperature of drying gas were 6 L/min and 350 degrees C, respectively. The optimized CE-MS method was successfully applied for the simultaneous determination of 12 nucleosides and nucleobases in natural and cultured Cordyceps. On the basis of quantitative results, the total content of nucleosides is much higher in cultured Cordyceps (9138+/-4823 microg/g for cultured C. sinensis; 3722+/-1446 microg/g for C. militaris) than in natural ones (2167+/-412 microg/g). However, the hypoxanthine (131+/-47 microg/g) and inosine (335+/-90 microg/g) are much higher in natural C. sinensis. Cordycepin, which is abundant in cultured C. militaris (2276.5+/-842.6 microg/g), is only found in natural C. sinensis with very low content and cannot be detected in the cultured ones.

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis.

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Study on the retention equation in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) is an effective technique for separating polar compounds. But its retention equation has not been studied systematically yet. In this study, an appropriate retention equation was established by using eight nucleosides as model analytes and by comparing four retention models on six different HILIC columns. As a result, retention equation Ink = a + b x InC(B) + cxC(B) could be quantitatively described the retention factors with good accuracy in HILIC mode. Based on this equation, the retention times of eight nucleosides under the conditions by other mobile phase can be predicted on each column. All of the predicted relative errors of retention times were smaller than 5%. The established retention model was also successfully applied to predict retention times of the real Traditional Chinese Medicine-Carthaus tinctorius L. sample.

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.

Enantioseparation of new nucleoside analogs, related to d4T and acyclovir, by chiral capillary electrophoresis using highly sulfated beta-cyclodextrins.

Baseline separation of some new acyclic nucleosides which are potential antiviral agents was achieved using cyclodextrin capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries, which were dynamically coated with polyethylene oxide (PEO). Operational parameters including (i) the nature and concentration of the chiral selectors, (ii) organic modifiers, (iii) temperature, and (iv) applied voltage were investigated. The use of charged CDs provides (i) a supplementary driving force for the compounds in a running buffer and (ii) enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-CD was found to be the most effective complexing agent and allowed good enantiomeric resolution. The complete resolution of five nucleoside analogs was obtained using 25 mM phosphate buffer, pH 2.5, containing either highly S-alpha-CD, S-beta-CD or S-gamma-CD at 30 degrees C with an applied field of 0.30 kV/cm. The apparent association constants of the inclusion complexes were calculated. The enantiomer migration order for the molecules investigated was determined and the detection limit of enantiomeric impurities was found to vary between 0.34 to 3.56 ng.mL(-1) for the first enantiomer.

Chromatographic separation of nucleosides using a cross-linked xylose isomerase crystal stationary phase.

Cross-linked xylose isomerase (EC 5.3.1.5., from Streptomyces rubiginosus) crystals (CLXIC) packed into a 7.8 x 300 mm steel column showed specific affinity towards uridine (Urd), cytidine (Cyd), adenosine (Ado), guanosine (Guo), and thymidine. These nucleosides eluted out of the CLXIC column in the same order as the corresponding nucleoside bases, indicating that the retention depends mainly on the base component of the molecule. The interaction of nucleosides with the CLXIC material was not based merely on ion exchange or hydrophobic interactions but also on the unique properties of the CLXIC column. Decrease in temperature increased the retention but not the resolution factors of the adjacent nucleosides. The CLXIC column maintained its separation capacity even when 100 mg of ribonucleosides in equimass amounts were injected into the column in a volume of 1 mL corresponding to 10% of the total column volume. Analysis of sugar beet molasses, a side stream from sucrose production, showed it to contain 1-2.5 mg mL(-1) of Urd, Cyd, Ado, and Guo. The CLXIC column was able to separate and enrich these nucleosides also from highly viscous sugar beet molasses. The CLXIC column was especially efficient in the purification of guanosine. Other commercially interesting sugar beet molasses components such as the acidic compounds betaine, gamma-amino butyric acid, and D- and L-pyroglutamic acids or neutral sucrose did not interact with the CLXIC material.

Identification and quantification of base and nucleoside markers in extracts of Ganoderma lucidum, Ganoderma japonicum and Ganoderma capsules by micellar electrokinetic chromatography.

The present paper describes the development of a micellar electrokinetic chromatographic method for the determination of nucleoside (adenosine, uridine) and base (uracil) markers in aqueous extracts of Ganoderma medicinal preparations. The markers were successfully separated within 10 min using an 80 mM borate buffer, with 25 mM sodium dodecyl sulfate adjusted to pH 9.0, an operating voltage of 22 kV, temperature of 20 degrees C and a hydrodynamic injection time of 5 s. Separations were carried out in a fused-silica capillary with peak detection by direct UV at 254 nm. Following semi-validation of the method, with each analyte showing a good linear relationship over a 0.2 to 20 ppm concentration range (correlation coefficients from 0.9986 to 0.9998), the amounts of the three markers in the various forms of Ganoderma were easily determined using a relatively simple extraction procedure.