Managing the column equilibration time in hydrophilic interaction chromatography.

For a wide variety of hydrophilic interaction chromatography stationary phases, a repeatable partial equilibration was demonstrated in gradient elution after purging with as little as 12 column volumes of mobile phase. Relative standard deviations of retention time of on average ~0.15% could be obtained after 1 or 2 conditioning (blank) runs. The equilibration period must be kept strictly constant, otherwise selectivity changes occur, but this is not problematic on modern instruments. Partial equilibration was largely independent of stationary phase or gradient slope. Alternatively, full column equilibration is favoured for stationary phases that do not trap extensive water layers, and for materials with a wider pore size that have a lower surface area. Temperatures somewhat above ambient also shorten the equilibration time. Some stationary phases under optimum conditions can achieve full column equilibration using purging with ~12 column volumes, which is useful for rapid set-up of isocratic separations or for conventional gradient analysis.

A new isoflavane-4-ol derivative from Melilotus officinalis (L.) Pall.

A new isoflavane derivative, melilofficinaside together with seven other metabolites including coumarin, uridine, methyl-α-d-fructofuranoside, and flavonoid glucosides were isolated from the aerial parts of Melilotus officinalis (L.) Pall.

Uridine derivatives from the seeds of Lepidium apetalum Willd. and their estrogenic effects.

Ten uridine derivatives (lepidiumuridine B-K) were isolated from the seeds of Lepidium apetalum Willd. Lepidiumuridine B-J were previously undescribed compounds, and were structurally characterized using analysis of their NMR and MS data. Lepidiumuridine C, D, I, and J increased cell proliferation and expression of ERα in the MCF-7 cell line. In addition, blockage of ERα completely abolished cell proliferation and expression of ERα in MCF-7 cells, suggesting that the proliferation effects of lepidiumuridine C, D, I, and J were ERα-mediated. The uridine derivatives might belong to undescribed phytoestrogens.

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.

The nucleoside uridine isolated in the gas phase.

Herein we present the first experimental observation of the isolated nucleoside uridine, placed in the gas phase by laser ablation and characterized by Fourier transform (FT) microwave techniques. Free from the bulk effects of their native environments, anti/C2'-endo-g+ conformation has been revealed as the most stable form of uridine. Intramolecular hydrogen bonds involving uracil and ribose moieties have been found to play an important role in the stabilization of the nucleoside.

Isolation of a new disaccharide nucleoside from Helleborus caucasicus: structure elucidation and total synthesis of hellecaucaside A and its ß-anomer.

Hellecaucaside A, a new disaccharide nucleoside featuring a 2'-O-α-D-ribofuranosyluridine skeleton and a 4-hydroxybenzoyl group at the 5' position, was isolated from the underground part of Helleborus caucasicus. The structure of the compound was elucidated by means of chemical degradation and spectroscopic analyses, such as 1D/2D NMR, chiral-GC, and HRMS. The total synthesis of hellecaucaside A and its ß-anomer was accomplished, unequivocally confirming the structure of the natural product.

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.

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.

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.

[Isolation and structure elucidation of chemical constituents from Pinellia ternata].

OBJECTIVE: To isolate and identify the chemical constituents of ethanol extract of Pinellia ternata. METHODS: The constituents were isolated by silica-gel, Sephadex LH-20 chromatography and HPLC techniques. The structures were identified by spectroscopic analysis including 2D NMR techniques and chemical properties. RESULTS: Nine compounds were obtained and identified as uridine (1), 5'-S-methyl-5'-thioadenosine (2), adenine (3), chrysophanol (4), 5-hydroxymethylfurfural (5), nicotinamide (6), (2S)-1-O-(9Z, 12Z-octadecadienoyl)-3-O-beta-galactopyranosylglycerol (7), daucosterol (8), beta-sitosterol (9). CONCLUSION: Compounds 2, 6, 7 are isolated from this plant for the first time.

Hypotensive and cardio-chronotropic constituents of Tinospora crispa and mechanisms of action on the cardiovascular system in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for the control of blood pressure. We previously found that an extract of Tinospora crispa stems decreased the mean arterial blood pressure (MAP) with a transient decrease, followed by an increase in the heart rate (HR) in rats. AIM OF THE STUDY: To identify the active components of the Tinospora crispa extract and investigate the mechanisms of action on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: The active components of Tinospora crispa extract were separated by column chromatography and a preparative HPLC. The effects and mechanisms of the active compounds on blood pressure and heart rate were studied in anesthetized, normal and reserpinized rats in vivo. RESULTS: 5 active compounds: adenosine, uridine, salsolinol, higenamine and tyramine were isolated. Adenosine decreased MAP and HR and this effect was inhibited by DMPX (A(2A) adenosine receptor antagonist). Uridine increased MAP and decreased HR and this was inhibited by suramin but not by DMPX. Salsolinol decreased the MAP and HR and this was inhibited by phentolamine but not by ICI-118,551 (ß(2)-adrenoceptor antagonist) or atropine. In reserpinized rats, salsolinol had a hypertensive effect that was inhibited by prazosin and phentolamine, but not by atenolol, and caused an increase in HR that was inhibited by atenolol, but not by prazosin or phentolamine. Higenamine decreased the MAP with an increase in HR. The hypotensive effect was inhibited by ICI-118,551 or atenolol, whereas the increase in HR was not inhibited by ICI-118,551. Atenolol inhibited the increase in HR at a small dosage of higenamine but potentiated it at a higher dosage. In reserpinized rats, a small dosage of higenamine tended to potentiate the effect but at a higher dosage it caused inhibition. ICI-118,551 significantly inhibited this hypotensive effect. Tyramine caused an increase in MAP and HR and these effects almost disappeared in reserpinized rats. CONCLUSIONS: The results demonstrate that these 5 compounds from Tinospora crispa acted in concert on the cardiovascular system of anesthetized rats. Salsolinol, tyramine and higenamine acted via the adrenoreceptors, whereas uridine and adenosine acted via the purinergic adenosine A(2) and P(2) receptors to decrease blood pressure with a transient decrease of HR followed by an increase.

The development of an evaluation method for capture columns used in two-dimensional liquid chromatography.

Capture columns are important interface tools for on line two-dimensional liquid chromatography (2D-LC). In this study, a systematic method was developed to evaluate and optimize the capture ability of capture columns by off-line method. First, the parameter Δt(R) (Δt(R)=t(2)-t(1)-t(0)-W) was introduced to quantitatively represent the capture ability of the capture column by connecting a capture column behind the first dimensional column. Based on the value of Δt(R), an appropriate capture column was selected after the first dimensional column was fixed. Then, the capture ability of the selected column was promoted by adjusting the mobile phase of the first dimensional column. Capture ability was also optimized using complex sample analysis software system (CSASS) software. Second, the elution mode of the trapped compounds on the capture column was investigated by connecting the capture column before the second dimensional column. More specifically, in mode I, capture column was connected to the second dimension without changing the flow rate direction and the trapped compounds must pass through the capture column and be eluted into the second dimensional column. The contrary connection mode was mode II. It was found that mode I is more suitable method for 2D-LC. Finally, an off-line reversed-phase/hydrophilic interaction liquid chromatography two-dimensional liquid chromatography (RP/HILIC 2D-LC) system with a C18 capture column was developed to demonstrate the practical application of this method.

[Studies on the chemical constituents from aerial parts of Gynura divaricata].

OBJECTIVE: To study the chemical constituents from aerial parts of Gynura divaricata. METHODS: The constituents were isolated on silica gel column chromatography, preparative TLC and Sephadex LH-20 column chromatography, identified by physicochemical properties and the structures were elucidated by spectral analysis. RESULTS: 10 compounds were isolated and identified as 2-(1', 2', 3', 4'-tetrahydroxybutyl)-6-(2", 3", 4"-trihydroxybutyl)-pyrazine (1), 2-(1', 2', 3', 4'-tetrahydroxybutyl)-5-(2", 3", 4"-trihydroxybutyl) -pyrazine (2), nicotinic acid (3), 5-hydroxy-picolinic acid(4), methyl-5-hydroxy-2- pyridinecarboxylate (5), adenosine (6), uridine (7), stigmasterol-5-O- beta-D-glucoside (8), dibutyl terephthalate (9), methyl chlorogenate (10). CONCLUSION: Compounds 1, 2, 5, 9, 10 are obtained from this genus for the first time, Compounds 3, 4 are obtained from this plant for the first time.

[Study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products].

OBJECTIVE: To study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products. METHODS: To determinate 13 samples of Cordyceps sinensis and its similar products by HPLC, and analyze the HPLC results with similar appraisal method and graphical methods of multivariate sample in two dimensional plane such as the methods of profile, radar chart and constellation graph. RESULTS: The similar appraisal method might synthesize the similar degree in quantification, while the graphical methods such as profile graph, radar chart and constellation graph could show more details about the classification and the characteristic of varieties directly. CONCLUSIONS: We recommend the combined application of similar appraisal method and the graphical methods due to its advantages on the judgment and characteristic analysis of fingerprint.

[Studies on the chemical constituents of the roots of Anemone altaica].

OBJECTIVE: To investigate the chemical constituents of the roots of Anemone altaica Fisch. ex C. A. May. METHODS: The constituents of n-BuOH-soluble portion were isolated and purified by means of chromatography. Compounds were identified by their physical characteristics and spectral features. RESULTS: Six compounds were isolated and identified as cimigenol-3-O-beta-D-xylopyranoside (1), cimigenol-3-O-beta-D-xylopyranol (1 -->3)-beta-D-xylopyranoside (2), isolariciresinol-9-O-beta-D-glucopyranoside (3), adenosine (4), uridine (5) and methyl-beta-D-glucopyranoside (6). CONCLUSION: All compounds are isolated from this genus for the first time.

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.

Sansanmycins B and C, new components of sansanmycins.

Two new antibiotics, sansanmycins B and C, were isolated from Streptomyces sp. SS. Their structures were elucidated by extensive 1D and 2D NMR and MS spectral analyses. Sansanmycins B and C exhibited inhibitory activity against our test strain of Pseudomonas aeruginosa with MIC values of 8.0 and 16 microg/ml, respectively. Sansanmycin B also exhibited inhibitory activity against Mycobacterium tuberculosis H37Rv and multidrug-resistant Mycobacterium tuberculosis, with the MIC values ranging from 8.0 to 20 microg/ml.

[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.

Kipukasins, nucleoside derivatives from Aspergillus versicolor.

Seven new aroyl uridine derivatives (kipukasins A-G; 1-7) were isolated from solid-substrate fermentation cultures of two different Hawaiian isolates of Aspergillus versicolor. The structures of compounds 1-7 were determined by analysis of NMR and MS data. The nucleoside portion of lead compound 1 was assigned as uracil-1-beta-D-ribofuranoside by spectral comparison with an authentic standard. The bioactivity of the original A. versicolor extracts was accounted for mainly by the presence of the known metabolite sterigmatocystin, but kipukasins A and B showed modest activity in assays against Gram-positive bacteria.