Identification of the major vanilloid component in Capsicum extract by HPLC-EC and HPLC-MS

A sensitive multi-channel HPLC-electrochemical (EC) method has been developed to determine the vanilloid content in the complex Capsicum annuum extract Capsibiol. Chromatographic separation was achieved within 10 min using a YMC Basic S5 column with a mobile phase containing chloroacetic acid, heptane sulphonic acid and acetonitrile. The multi-channel detector simultaneously applied four potentials between +500 and +800 mV (referenced to a silver/silver chloride electrode) to four glassy carbon working electrodes. The most abundant (0.94 mg/g) vanilloid analogue in the Capsibiol sample demonstrated an electrochemical reactivity and retention time similar to that of vanillic acid in HPLC-EC analysis. Its identity was confirmed by HPLC-MS using a Zorbax SB-CN column with a mobile phase containing formic acid and methanol.     

Patterns of Genetic Variation in Swertia przewalskii, an Endangered Endemic Species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Φ _ST comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory θ_B) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.     

A validated stability-indicating HPLC method for the determination of PEGylated puerarin in aqueous solutions

The aim of this study was to develop a validated specific stability-indicating HPLC method for the quantitative determination of PEGylated puerarin (PEG-PUE) in aqueous solutions. The method was validated by subjecting PEG-PUE to forced degradation under stress conditions of acid, alkali, water hydrolysis, and oxidation. Both PEG-PUE and puerarin (PUE) were simultaneously determined and separated on CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1.0 mL min⁻¹ and detection wavelength was set at 250 nm. Both calibration curves showed good linear regression (r ≥ 0.9998) within test ranges. The LOD and LOQ of PEG-PUE were determined to be 3 and 9 μg mL⁻¹ respectively. Degradation of PEG-PUE followed pseudo-first-order kinetics with t_1/2 of 59 min at pH 9.0 and 17.79 h at pH 7.4. However, at pH 5.0 and 2.0, there was no significant degradation of PEG-PUE over time. In conclusion, the method was observed to have the necessary specificity, precision, and accuracy, and to be suitable for quantity monitoring the degradation process of PEG-PUE during stability studies. The degradation studies may give insight into useful information for formulation development of PEG-PUE.     

Analysis of macamides in samples of Maca (Lepidium meyenii) by HPLC-UV-MS/MS

The macamides are a distinct class of secondary metabolites that have so far been found only in Lepidium meyenii Walp. (Maca). Using HPLC-UV-MS/MS, the main macamides have been identified as n-benzylhexadecanamide, n-benzyl-(9Z)-octadecenamide, n-benzyl-(9Z, 12Z)-octadecadienamide, n-benzyl-(9Z, 12Z, 15Z)-octadecatrienamide and n-benzyloctadecanamide. The identities of n-benzyl-(9Z)-octadecenamide and n-benzyl-(9Z, 12Z)-octadecadienamide were confirmed by comparison of chromatographic and spectral properties with synthetic analogues. Total macamides have been quantified by HPLC-UV in plant material from different vendors using n-benzylhexadecanamide as an external standard. The amount of macamides in the dried plant material ranged from 0.0016 to 0.0123%.     

Quantification of Aconitum alkaloids in aconite roots by a modified RP-HPLC method

The three Aconitum alkaloids, aconitine (1), mesaconitine (2) and hypaconitine (3), are pharmacologically active but also highly toxic. A standardised method is needed for assessing the levels of these alkaloids in aconite roots in order to ensure the safe use of these plant materials as medicinal herbs. By optimising extraction, separation and measurement conditions, a reliable, reproducible and accurate method for the quantitative determination of all three Aconitum alkaloids in unprocessed and processed aconite roots has been developed. This method should be appropriate for use in the quality control of Aconitum products. The three Aconitum alkaloids were separated by a modified HPLC method employing a C18 column gradient eluted with acetonitrile and ammonium bicarbonate buffer. Quantification of Aconitum alkaloids, detected at 240 nm, in different batches of samples showed that the content of 1, 2 and 3 varied significantly. In general, the alkaloid content of unprocessed roots was higher than that of processed roots. These variations were considered to be the result of differences in species, processing methods and places of origin of the samples.     

Inhibition of the onset of vitellogenesis by abnormal retention of mature eggs in the stick insect,Carausius morosus

Mature eggs stored in the lateral oviducts are unusual in Carausius morosus. When egg laying is experimentally prevented, ovarian production is inhibited by the accumulation of mature eggs within the lateral oviducts. Such storage affects only the very small terminal oocytes and not the vitellogenic follicles, which attain to term and are ovulated. The fact that unilateral retention of eggs affects only the corresponding ovary strongly suggests that the mature eggs themselves inhibit ovarian production in the stick insect without the involvement of an endocrine organ. It is likely that this mode of action, different from that suggested for the housefly or Rhodnius prolixus, is correlated with the independence of ovarian development from the corpus allatum hormone in the stick insect.     

Determination of the triacylglycerol composition of coffee beans by reverse-phase high-performance liquid chromatography

Reverse-phase HPLC with refractive index and light scattering detectors in isocratic and gradient elution modes, respectively, was applied for the separation of the major triacylglycerols (TAG) in coffee lipids. Twelve TAG species could be identified and determined using a linear gradient of acetonitrile in dichloromethane: dichloroethane. The quantitative evaluation was based on the relative area percentages derived directly from a data-station. The procedure was applied to determine the TAG composition of three types of coffee beans harvested in two coffee producing areas in Brazil and dried by two commonly used procedures. No significant differences in the TAG compositions due to the type, origin and drying procedure were found.     

Quantitative determination of Aconitum alkaloids in blood and urine samples by high-performance liquid chromatography

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.     

9种杨属植物雄花序中黄酮类含量的HPLC法测定

杨树(杨柳科Ｓａｌｉｃａｃｅａｅ杨属ＰｏｐｕｌｕｓＬ.植物)树皮、雄花序、芽脂、叶等均可药用。1977年版《中国药典》收载加杨(Ｐｏｐｕｌｕｓ×ｃａｎａｄｅｎｓｉｓＭｏｅｎｃｈ)、毛白杨(ＰｏｐｕｌｕｓｔｏｍｅｎｔｏｓａＣａｒｒ.)及杨属其他植物的雄花序,用于治?..     

Quantification of tagitinin C in Tithonia diversifolia by reversed-phase high-performance liquid chromatography

A simple, rapid and reliable reversed-phase high-performance liquid chromatographic method for the determination of tagitinin C, an anti-plasmodial sesquiterpene lactone isolated from the aerial parts of Tithonia diversifolia, has been developed. The assay has been used to quantify tagitinin C in various extracts of the aerial parts of T. diversifolia.     

Synchronization by light of the circadian rhythm of motor activity in the crayfish

Abstract

We have studied the pattern for resetting the circadian rhythm in the spontaneous motor activity of the crayfish. Spontaneous motor activity was recorded continously at a constant temperature and under free running conditions in complete darkness. The effect of single light pulses applied at different circadian times, on the circadian rhythm of motor activity was measured in both transient stage and steady state. The results led us to construct a phase‐transition curve and phase‐response curve which were analyzed to obtain information about the oscillators which underlie the circadian rhythm of motor activity.     

Enhanced chemiluminescence in the oxidation of luminol and an isoluminol cortisol conjugate by hydrogen peroxide in reversed micelles

The chemiluminescent oxidation of luminol and an isoluminol cortisol conjugate (ABICOR) by hydrogen peroxide has been studied in cetyltrimethylammonium bromide (CTAB) reversed micelles in octane-chloroform (1 : 1). The maximum chemiluminescence intensity of both compounds is dependent on the initial concentrations of the H₂O₂ and substrates, the pH value of the micelle polar phase and the H₂O/CTAB ratio. The optimum pH ranged from 8.5 to 9.5. Under comparable conditions, the chemiluminescence intensity for luminol was 15-fold higher than for the ABI-COR conjugate. A mechanism of oxidation of the substrates in reversed micelles is proposed and the possible mechanisms of inhibition by the substrate and oxidant is discussed.     

Triterpenes in the hexane extract of leaves of Olea europaea L.: analysis using 13C-NMR spectroscopy

Two neutral triterpenes and a triterpene acid were identified and quantified directly, in the absence of any purification steps, in a precipitate obtained during the industrial extraction of the leaves of Olea europaea L. using 13C-NMR spectroscopy (spectrometer operating at 4.7 T equipped with a 10 mm probe). The method was optimised in order to reduce the duration of analysis with a routine NMR spectrometer. Together with long-chain linear compounds, erythrodiol, uvaol and oleanolic acid accounted for 27.3, 18.3 and 12.5% of the precipitate, respectively.     

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature

Quantitative real‐time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species‐specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial‐feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host.     

Computer-assisted, high-performance liquid chromatography with mass spectrometric detection for the analysis of coumarins in Peucedanum palustre and Angelica archangelica

A reversed-phase HPLC method with atmospheric pressure chemical ionisation MS detection has been developed for the separation and identification of coumarins in plants of Peucedanum palustre L. (Moench) and Angelica archangelica (L.) var. archangelica. The Turbo Method Development program was utilised to optimise the mobile phase with two organic solvents (acetonitrile and methanol) and two aqueous solutions (1.0% formic acid and 10 mM ammonium acetate). Optimisation of the solvent gradients for the method was performed with the aid of the DryLab program. Analyses were carried out using a Phenomenex Prodigy RP C18 column. Fifty-two peaks (14 of which were associated with coumarins) were separated in 30 min from extracts of P. palustre, and 48 peaks (15 associated with coumarins) from extracts of A. archangelica. A total of 21 different coumarin-type compounds were identified in the aerial and the underground parts of the title plants. Isopimpinellin and pimpinellin were found for the first time in P. palustre and were identified by comparison of retention times and MS data obtained following the analysis of pure standards. This is the first report of the coumarin composition of the umbels of P. palustre.     

Alternative splicing factor or splicing factor-2 plays a key role in intron retention of the endoglin gene during endothelial senescence

Alternative splicing involving intron retention plays a key role in the regulation of gene expression. We previously reported that the alternatively spliced short isoform of endoglin (S-endoglin) is induced during the aging or senescence of endothelial cells by a mechanism of intron retention. In this work, we demonstrate that the alternative splicing factor or splicing factor-2 (ASF/SF2) is involved in the synthesis of endoglin. Overexpression of ASF/SF2 in endothelial cells switched the balance between the two endoglin isoforms, favoring the synthesis of S-endoglin. Using a minigene reporter vector and RNA immunoprecipitation experiments, it was shown that ASF/SF2 interacts with the nucleotide sequence of the endoglin minigene, suggesting the direct involvement of ASF/SF2. Accordingly, the sequence recognized by ASF/SF2 in the endoglin gene was identified inside the retained intron near the consensus branch point. Finally, the ASF/SF2 subcellular localization during endothelial senescence showed a preferential scattered distribution throughout the cytoplasm, where it interferes with the activity of the minor spliceosome, leading to an increased expression of S-endoglin mRNA. In summary, we report for the first time the molecular mechanisms by which ASF/SF2 regulates the alternative splicing of endoglin in senescent endothelial cells, as well as the involvement of ASF/SF2 in the minor spliceosome.     

Sensitivity improvement of circular dichroism detection in HPLC by using a low-pass electronic noise filter: Application to the enantiomeric determination purity of a basic drug

The quality control of chiral drugs requires the determination of their enantiomeric purity. Nowadays, circular dichroism (CD) spectroscopy is gaining increasing importance in pharmaceutical analysis because of the commercially available CD detector in liquid chromatography. The separation of the two enantiomers of a basic drug (efaroxan) was achieved by high performance liquid chromatography using an amylose-derivated column with both UV and CD detections. A baseline-resolved separation (resolution: 5) was obtained after optimization of the mobile phase composition with hexane-ethanol-diethylamine (90:10:0.05; v/v/v). The use of a commercial low-pass electronic noise filter of the CD signal has improved the signal-to-noise ratio by a factor twelve and allowed the quantitation of each enantiomer in the 1.25-300 microg ml(-1) concentration range. The CD linear calibration curve, expressed in terms of stereoisomer height ratio versus concentration ratio, was plotted over the 0.4-6% range. A correlation coefficient greater than 0.999 was obtained by least-squares regression and the limit of detection for the distomer/eutomer ratio was estimated at 0.14%. Although the method validation showed good repeatability on the retention times (RSD < 0.9%), on the peak height ratios (RSD < 8.7%) of each enantiomer only up to 99.2% enantiomeric purity was achieved.