Reversed-phase High-performance Liquid Chromatography Analysis Research Articles

Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis.

Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm.

Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process.

An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis.

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N-Acetylmuramidase Activity.

Bacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novel Salmonella bacteriophage endolysin, Gp110, which comprises an uncharacterized domain of unknown function (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents. We report the functional and biochemical characterization of the Salmonella phage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 has N-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

PHENOLIC COMPOSITION AND BIOLOGICAL (ANTI DIABETIC AND ANTIOXIDANT) ACTIVITIES OF DIFFERENT SOLVENT EXTRACTS OF AN ENDEMIC PLANT (HELIOTROPIUM STRIGOSUM)

Heliotropium strigosum plant is widely used in traditional medicines for treatment of various ailments. In current study, an effort was made to evaluate phenolic profile as well as antidiabetic and antioxidant activities of crude methanol extract and different solvent (n-hexane, ethyl acetate, chloroform and aqueous) fractions of H. strigosum . Total phenolic contents were determined by spectrophotometric assay. Polyphenolic compounds in crude methanol extract and each solvent fraction were identified by reverse phase high performance liquid chromatography (RP-HPLC). Antioxidant and antidiabetic activities were determined using DPPH and α-amylase inhibition assays, respectively. The outcome of spectrophotometric assay showed that methanol extract had higher amount of total phenolic (84.50 ± 0.06 μg GAE/ mg of plant extract) contents than aqueous, ethyl acetate, chloroform and n-hexane fractions, respectively. The RP-HPLC analysis revealed the maximum number of phenolic (chromotropic acid, quercetin, trans-4-hydroxy-3-methoxy cinnamic acid, vanillic acid, gallic acid, caffeic acid, m-coumaric acid, p-coumaric acid, syringic acid, sinapic acid and ferulic acid) compounds in methanol extract. Methanol extract (IC 50 = 8.97 μg/mL) exhibited the maximum antidiabetic activity followed by aqueous (IC 50 = 20.04 μg/mL), ethyl acetate (IC 50 = 27.79 μg/mL), chloroform (IC 50 = 56.87 μg/mL) and hexane (IC 50 = 32.16 μg/mL) fractions, respectively. The outcome of antioxidant assay revealed that methanol extract was the leading one with regard to antioxidant activity at different doses (10 to 250 μg/mL). The current study concludes that H. strigosum solvents extracts with significant phenolic profile and potent biological activities could be explored for potential uses in neutraceutical and pharmaceutical industries.

An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/β-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

Resolution of Rac-Bambuterol via Diastereoisomeric Salt Formation with o-Chloromandelic Acid and Differences in the Enantiomers' Pharmacodynamical Effects in Guinea Pigs and Beagles.

In this study an enantioseparation method for rac-bambuterol (5-(2-(tert-butylamino)-1-hydroxyethyl)-1,3-phenylene bis(dimethylcarbamate)) via diastereoisomeric salt formation with o-chloromandelic acid was developed. The enantiomeric excess (ee) values and chemical purities of the desired products were confirmed by high-performance liquid chromatography (HPLC) using chiral stationary phase and reverse-phase HPLC analyses, respectively. The ee values and the chemical purities both exceeded 99%. Animal experiments showed that (R)-bambuterol was a potent inhibitor for histamine-induced asthma reactions. (S)-bambuterol was ineffective in relaxing the airways. Both enantiomers increased heart rates in beagles. Therefore, replacing rac-bambuterol with (R)-bambuterol could be beneficial for asthma patients.

Optimizing Labeling Conditions for Cysteine-Based Peptides with99mTc

Radiolabelled peptides have attracted a great deal of attention due to their wide applicability in the development of target-specific radiopharmaceuticals. They can easily be used in diagnostic imaging as carriers for the delivery of radionuclides to tumors as well as for therapy. Previous investigations revealed that technetium(V) could form stable complexes with peptide-based ligands of N3S type such as Cys-Gly-Gly-Gly. Herein, a targeting HER-2 receptor peptide was labeled with technetium-99m (99mTc) with two different types of tetrapeptide-based ligands, Cys-Gly-Gly-Gly and Cys-Ser-Ser-Ser. The effect of experimental parameters in the labeling procedure such as type of buffer solutions, pH of media, and type of exchange ligands were optimized toward obtaining maximum labeling yield. The optimum labeling conditions were different for two peptides. Shelf life of both labeled peptides was determined by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and thin layer chromatography (TLC) that showed radiochemical yield up to 95% even after 4 h.

A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production.

A novel chromatic visible screening method using bromothymol blue (BTB) as a color indicator and cetylpyridinium chloride (CPC) as a mediator was constructed to obtain the high titer surfactin-producing strains. The reliability and quantification accuracy of color shift were also confirmed. Regular chromatic responses from faint yellow-green to dark green and bright blue reflected the different ranges of surfactin concentrations. Moreover, the quantitative accuracy of surfactin quantification in the range of 100-500mg/L was verified by reverse-phase high-performance liquid chromatography (RP-HPLC) using different fermentation supernatant samples. Using this CPC-BTB method, a superior surfactin producer, Bacillus subtilis THY-15, was successfully screened. The producer's surfactin (Srf) titer reached 1240mg/L. RP-HPLC analysis of THY-15 revealed four surfactin isoforms. As identified by amino acid analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, the isoforms of surfactin in fraction 1, 2 and 4 had the same circular peptide sequence of Glu-Leu-Leu-Val-Asp-Leu-Leu but different iso-C13, C14 and C15 fatty acid chains, but the isoform in fraction 3 possessed a special peptide sequence of Glu-Val-Leu-Leu-Asp-Leu-Val.

Phenolic Compounds and Bioactivity of Healthy and Infected Grapevine Leaf Extracts from Red Varieties Merlot and Vranac (Vitis vinifera L.).

We investigated the phenolic composition, radical scavenging activity, and antimicrobial activity of grapevine leaf extracts from two red grape varieties, Vranac and Merlot (Vitis vinifera L.). The extracts were prepared from healthy grapevine leaves and those infected by Plasmopara viticola (downy mildew). The phenolic composition of the grapevine leaf extracts was determined using spectrophotometric assays and reverse-phase high performance liquid chromatography (RP-HPLC). The radical scavenging activity of grapevine leaf extracts was determined by the 2,2-diphenyl-1-picrylhydrazyl assay, and their antimicrobial activity was determined by microwell dilution tests. The total phenolic content was higher in healthy grapevine leaf extracts than in infected grapevine leaf extracts. The RP-HPLC analysis detected significant amounts of flavonols, phenolic acids, and flavan-3-ols, and small amounts of stilbenes in the grapevine leaf extracts. Compared with the infected grapevine leaf extracts, the healthy grapevine leaf extracts were richer in flavonols, phenolic acids, and flavan-3-ols, but had lower stilbenes contents. All extracts showed strong free radical scavenging activity, which was strongly correlated with the total phenolic content (R(2) = 0.978). The extracts showed a stronger antimicrobial activity towards Gram-positive bacterial strains than towards Gram-negative bacterial strains and yeast. The phenolic compounds in grapevine leaves were responsible for their strong radical scavenging and antimicrobial activities. Together, these results demonstrate that grapevine leaves have high nutritional value and can be used as a fresh food and to prepare extracts that can be used as additives in food and medicines.

Optimization of Extraction Techniques and RP-HPLC Analysis of Antidiabetic and Anticancer Drug Mangiferin from Roots of ‘Saptarangi’ (Salacia chinensis L.)

Saptarangi (Salacia chinensis L.) is a chief source of mangiferin, which has antidiabetic and anticancer properties. Extraction using continuous shaking extraction (CSE), ultrasonic extraction (UE), microwave assisted extraction (MAE), and steam bath assisted extraction (SBAE) were evaluated for extraction of mangiferin (MF) from roots of S. chinensis. The extracts were analyzed by reverse phase-high performance liquid chromatography (RP-HPLC) and Fourier transform-infra red spectroscopy (FT-IR) analysis. Operational parameters such as different solvents and exposure period were also evaluated. N,N-Dimethyl formamide (DMF 30%, v/v) extracted higher quantity of MF compared to other solvents. The results shows that the extraction of MF by SBAE was more efficient followed by MAE, CSE, and UE methods. Maximum quantity of MF (391.80 ppm) was obtained by SBAE technique and DMF (30%) as a solvent system. SBAE is the easiest and best method for screening of MF from S. chinensis. Furthermore, use of FT-IR validates the RP-HPLC technique for identification and quantification.

Gac-mediated changes in pyrroloquinoline quinone biosynthesis enhance the antimicrobial activity of Pseudomonas fluorescens SBW25.

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In Pseudomonas fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors.

Liquid Chromatographic Method for Simultaneous Quantitation of Clopidogrel, Aspirin and Atorvastatin in Rat Plasma and Its Application to the Pharmacokinetic Study.

A simple and robust analytical reversed-phase high-performance liquid chromatography method was developed and validated for simultaneous chromatographic elution of three cardiovascular drugs, namely clopidogrel, aspirin (ASP) and atorvastatin. The method was developed in rat plasma and dosage formulation with high-quality chromatographic separation between the drug peaks by using a stainless steel analytical column thermo beta-basic, C18 (25 × 0.46 cm, 5 µm). The system was operated at 25°C using a mobile phase consisting of acetonitrile and phosphate buffer (pH 3.0) in the gradient ratio at a flow rate of 1 mL min(-1) with ultraviolet detection monitored at 232 nm. The parametric statistics, i.e., correlation coefficient of 0.999, was assessed for all the drugs having linearity over the tested concentration range (10-10,000 ng mL(-1)) in rat plasma using an unweighted calibration curve. The accuracy of samples for six replicate measurements at lower limit of quantitation level was within limit. The method was applicable for the quality control of the mentioned drugs in raw material, bulk drug and pharmaceutical formulations as well as in pharmacokinetic studies.

RP-HPLC and spectroscopic characterization of Suwannee River water NOM after concentrated urea treatment and dialysis

Suwannee River natural organic matter (SRNOM) was treated by 7M urea and then purified by dialysis on 10 kDa membrane. The untreated SRNOM and treated (USRNOM) samples were examined using UV–visible and fluorescence spectroscopies and reversed-phase high-performance liquid chromatography (RP-HPLC) with online absorbance and fluorescence detection. USRNOM was 1.5-fold more absorbing at 280nm than SRNOM and four fold less fluorescent than SRNOM upon excitation at 270 nm. RP-HPLC analyses of the two samples revealed that USRNOM was somewhat more hydrophobic than SRNOM and both samples contained at least two groups of HS-like fluorophores with different hydrophobicity and protein-like fluorophore(s). Data indicate that protein-like fluorophores were not lost during dialysis. They showed hydrophobic properties and seemed highly fluorescent. HS-like and protein-like fluorophores from water NOM could be successfully separated by RP-HPLC. This raises the prospect of their further research and identification and could be significant for future NOM chemical structure characterization.

Evaluation of phenolic extract of licorice roots in diets of Nile tilapia (Oreochromis niloticus)

Aims The aims of this study were to evaluate the effect of methanolic extract of licorice roots (MELRs) as a feed additive on the growth performance, feed utilization, and innate immunity of Nile tilapia ( Oreochromis niloticus) , and to screen the phenolic compounds contained in the different MELRs through reverse-phase high-performance liquid chromatography (HPLC). Background Licorice is doubtless one of the most popular medicinal plants, with its roots being the most used part. Particularly, the species Glycyrrhiza glabra L. has well-known therapeutic properties, which have been documented since the Egyptian age. Materials and methods MELR was prepared as follows: in a typical experiment, 500 g of the plant was dried at room temperature. The dried plant was defatted separately with n -hexane in a Soxhlet apparatus for 15 h. The plant residue was then extracted with methanol in a Soxhlet apparatus for 20 h. The obtained data were analyzed using one-way procedure SPSS according to the following model: Y ij = μ+ T i + e ij , where μ is the over mean, T i is the fixed effect of the rocket supplementation (1…4), and e ij is random error. The differences between the experimental groups were determined using Duncan's multiple range test. Results and discussion Screening the phenolic compounds contained in the different MELR achieved through reverse-phase HPLC analysis showed 21 phenolic and flavonoid compounds. The MELR supplementation enhanced fish growth compared with the control diet, and the highest fish performance was obtained at 0.04% MELR level. Supplementation with MELR was found to have an antibacterial activity antagonistic to pathogenic bacteria Aeromonas jandaei infection in fish. It is recommended that 0.04% MELR be used as a feed additive for Nile tilapia to enhance its growth performance, health, and innate immunity. Conclusion This study was carried out to evaluate the effect of MELRs as a feed additive on the growth performance, feed utilization, and innate immunity of Nile tilapia. MELR enhances fish growth rate, feed utilization, and innate immunity. The highest fish growth was obtained when fish were fed a diet containing 0.04% extract.

The Kinetic Stability of Cytochrome c Oxidase: Effect of Bound Phospholipid and Dimerization

Thermally induced transitions of the 13-subunit integral membrane protein bovine cytochrome c oxidase (CcO) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). Thermal denaturation of dodecyl maltoside solubilized CcO proceeds in two consecutive, irreversible, kinetically driven steps with the apparent transition temperatures at ∼ 51°C and ∼ 61°C (5μM CcO at scan rate of 1.5 K/min). The thermal denaturation data were analyzed according to the Lyubarev and Kurganov model of two consecutive irreversible steps. However, because of the limitation of the model to describe the complex mechanism of the thermal denaturation of CcO, the obtained results were utilized only for comparison purposes of kinetic stabilities of CcO under specific protein concentration (5μM) and scan rate (1.5 K/min). This enabled us to show that both the amphiphilic environment and the self-association state of CcO affect its kinetic stability. Kinetic stabilities of both steps are significantly decreased when all of the phospholipids are removed from CcO by phospholipase A2 (the half-life decreases at 37°C). Conversely, dimerization of CcO induced by sodium cholate significantly increases its kinetic stability of only the first step (the half-life increases at 37°C). Protein concentration-dependent nonspecific oligomerization also indicate mild stabilization of CcO. Both, reversed-phase high-performance liquid chromatography (HPLC) and SDS-PAGE subunit analysis reveal that the first step of thermal denaturation involves dissociation of subunits III, VIa, VIb, and VIIa, whereas the second step is less well defined and most likely involves global unfold and aggregation of the remaining subunits. Electron transport activity of CcO decreases in a sigmoidal manner during the first transition and this dependence is very well described by kinetic parameters for the first step of the thermal transition. Therefore, dissociation of subunit III and/or VIIa is responsible for temperature-induced inactivation of CcO because VIa and VIb can be removed from CcO without affecting the enzyme activity. These results demonstrate an important role of tightly bound phospholipids and oligomeric state (particularly the dimeric form) of CcO for kinetic stability of the protein.

Nanoencapsulation of a Withania somnifera extract with PCL and MPEG-PCL di-block copolymer

Withania sominifera (L) is a well-known Indian medicinal plant used to treat stress, arthritis insomnia and age related disorders including neurodegenerative disorders. It has several vernacular names including aswaganda and ginseng winter cherry [1 – 2]. Withanolides are the major and characteristic active compounds of this species [3]. The isolated fraction of withanolides have been studied for their anti-tumor, antiinflammatory, anti-stress, anti-oxidant, immuno-modulatory, cardio-protective and neuroprotective activity [4]. To enhance the solubility, bioavailability, protection from toxicity and increase the pharmacological activity of herbal extracts, several approaches has been proposed. Among them, plant extract based novel drug delivery system attracted much attention. In this investigation, we have developed and characterized a purified W. somnifera extract (WSE) encapsulated in PCL and MPEG-PCL nanoparticles and identified the entrapped compounds by reverse phase high performance liquid chromatography (RP-HPLC). WSE was obtained from leaf biomass extraction with aqueous MeOH and further purification with dichloromethane. MPEG-PCL di-block polymer was synthesised by ring opening polymerization of ε-Caprolactone on MPEG using Sn(Oct)2 as catalyst. Prepared WSE-nanoparticles were characterised by laser doppler anemometry and Transmission electron microscopy (TEM). The results from TEM and laser doppler anemometry confirmed that the size of PCL-WSE and MPEG-PCL-WSE nanoparticles ranged between 220 – 250nm and spherical in shape (Figure 1). The MPEG-PCL-WSE nanoformulation showed higher entrapment efficacy (54.4%) than PCL-WSE nanoparticles (31.2%). The RP-HPLC analysis revealed that Withanolide A, Withaferin-A, Withanolide-B were major compounds encapsulated in the nanoparticles.

Role of urinary cations in the aetiology of bladder symptoms and interstitial cystitis

To identify and characterise urinary cationic metabolites, defined as toxic factors, in patients with interstitial cystitis (IC) and in control subjects. To evaluate the cytotoxicity of the urinary cationic metabolite fraction of patients with IC vs control subjects and of individual metabolites in cultured urothelial cells. Cationic fractions (CFs) were isolated from the urine specimens of 62 patients with IC and 33 control subjects by solid-phase extraction. CF metabolites were profiled using C18 reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection, quantified by area-under-the-peaks using known standards, and normalized to creatinine. RP-HPLC and liquid chromatography (LC)-mass spectrometry (MS)/tandem MS (MS/MS) were used to identify major CF peaks. HTB-4 urothelial cells were used to determine the cytotoxicity of CFs and of individual metabolites with and without Tamm-Horsfall protein (THP). RP-HPLC analysis showed that metabolite quantity was twofold higher in patients with IC compared with control subjects. The mean (SEM) for control subjects vs patients was 3.1 (0.2) vs 6.3 (0.5) mAU*min/μg creatinine (P < 0.001). LC-MS identified 20 metabolites. Patients with IC had higher levels of modified nucleosides, amino acids and tryptophan derivatives compared with control subjects. The CF cytotoxicity was higher for patients with IC compared with control subjects. The mean (SEM) for control subjects vs patients was -2.3 (2.0)% vs 36.7 (2.7)% (P < 0.001). A total of 17 individual metabolites were tested for their cytotoxicity. Cytotoxicity data for major metabolites were all significant (P < 0.001): 1-methyladenosine (51%), 5-methylcytidine (36%), 1-methyl guanine (31%), N(4)-acetylcytidine (24%), N(7)-methylguanosine (20%) and L-Tryptophan (16%). These metabolites were responsible for higher toxicity in patients with IC. The toxicity of all metabolites was significantly lower in the presence of control THP (P < 0.001). Major urinary cationic metabolites were characterised and found to be present in higher amounts in patients with IC compared with control subjects. The cytotoxicity of cationic metabolites in patients with IC was significantly higher than in control subjects, and control THP effectively lowered the cytotoxicity of these metabolites. These data provide new insights into toxic factor composition as well as a framework in which to develop new therapeutic strategies to sequester their harmful activity, which may help relieve the bladder symptoms associated with IC.

Anti-cholinesterase activity of the standardized extract of Syzygium aromaticum L.

Background:Clove (Syzygium aromaticum) is a well-known culinary spice with strong aroma; contains a high amount of oil known as clove oil. The major phyto-constituent of the clove oil is eugenol. Clove and its oil possess various medicinal uses in indigenous medicine as an antiseptic, anti-oxidant, analgesic and neuroprotective properties. Thus, it draws much attention among researchers from pharmaceutical, food and cosmetic industries.Objective:The aim of the present study was to determine the anti-cholinesterase activity of the methanol extract of clove, its oil and eugenol.Materials and Methods:In vitro anti-cholinesterase activity of S. aromaticum was performed by a thin layer chromatography bio autography, 96 well micro titer plate and kinetic methods. Reverse phase high performance liquid chromatography (RP-HPLC) analysis was carried out to identify the biomarker compound eugenol in clove oil.Results:Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition study revealed that eugenol possess better inhibition of the enzymes than extract and oil. Clove extract, its oil and eugenol showed better inhibition of AChE than BChE. Polyphenolic compound eugenol was detected through RP-HPLC analysis. The content of eugenol in essential oil was found to be 0.5 μg/ml. Kinetic analysis of the cholinesterase inhibition study of the extract; clove oil and eugenol have shown that they possess mixed type of inhibition for AChE and non-competitive type of inhibition for BChE.Conclusion:These results might be useful in explaining the effect of clove as anti-cholinesterase agent for the management of cognitive ailments like Alzheimer's disease.

The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

Preparation of molecularly imprinted polymer for use as SPE adsorbent for the simultaneous determination of five sulphonylurea herbicides by HPLC

A high selective pre-treatment method for the analysis of sulphonylurea herbicides (SUHs) in rice grain samples based on molecularly imprinted solid-phase extraction (MISPE) was developed. The molecularly imprinted polymers (MIPs) were synthesised with high adsorption capacity and suitable particle size using pyrazosulphuron ethyl (PS) as the template and by screening three porogens and porogen volumes. The resulting MIPs showed high selectivity for PS. The MISPE procedure was developed for the purification and enrichment of PS, bensulphuron methyl (BSM), tribenuron methyl (TBM), metsulphuron methyl (MSM) and nicosulphuron (NS) from a rice grain sample prior to reversed-phase high-performance liquid chromatography (HPLC) analysis. Average recoveries of the PS, NS, MSM and BSM were 95.6%, 88.6%, 87.8% and 89.4%, respectively, but the recovery of TBM was lower (48.2%). This pre-treatment methodology for extracting SUHs was simple and cleaner extractions. The limits of detection ranged from 10.1 to 50.0ngL−1. Five local rice grain samples were analysed by HPLC–MISPE, and PS was detected in all five samples, while BSM was detected in one sample.