HPLC Peak Research Articles

An in vitro study of anti-inflammatory activity of standardised Andrographis paniculata extracts and pure andrographolide.

BackgroundThe anti-inflammatory activity of Andrographis paniculata (Acanthaceae), a traditional medicine widely used in Asia, is commonly attributed to andrographolide, its main secondary metabolite. Commercial A. paniculata extracts are standardised to andrographolide content. We undertook the present study to investigate 1) how selective enrichment of andrographolide in commercial A. paniculata extracts affects the variability of non-standardised phytochemical components and 2) if variability in the non-standardised components of the extract affects the pharmacological activity of andrographolide itself.MethodsWe characterized 12 commercial, standardised (≥30% andrographolide) batches of A. paniculata extracts from India by HPLC profiling. We determined the antioxidant capacity of the extracts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, oxygen radical antioxidant capacity (ORAC) and a Folin-Ciocalteu (FC) antioxidant assays. Their anti-inflammatory activity was assessed by assaying their inhibitory effect on the release of tumor necrosis factor alpha (TNF-α) in the human monocytic cell line THP-1.ResultsThe andrographolide content in the samples was close to the claimed value (32.2 ± 2.1%, range 27.5 to 35.9%). Twenty-one non-standardised constituents exhibited more than 2-fold variation in HPLC peak intensities in the tested batches. The chlorogenic acid content of the batches varied more than 30-fold. The DPPH free radical scavenging activity varied ~3-fold, the ORAC and FC antioxidant capacity varied ~1.5 fold among batches. In contrast, the TNF-α inhibitory activity of the extracts exhibited little variation and comparison with pure andrographolide indicated that it was mostly due to their andrographolide content.ConclusionsStandardised A. paniculata extracts contained the claimed amount of andrographolide but exhibited considerable phytochemical background variation. DPPH radical scavenging activity of the extracts was mostly due to the flavonoid/phenlycarboxylic acid compounds in the extracts. The inhibitory effect of andrographolide on the release of TNF-α was little affected by the quantitative variation of the non-standardised constituents.

Simultaneous detection of five pathogenic Vibrio species in seafood by a multiplex polymerase chain reaction coupled with high performance liquid chromatography assay

A multiplex polymerase chain reaction-based procedure followed by high performance liquid chromatography (mPCR-HPLC) assay was developed for the simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus and Vibrio mimicus from seafood. A set of multiplex PCR primers was designed based on the dnaJ gene of Vibrio species and was employed in a multiplex PCR procedure, then PCR products were subjected to HPLC analysis with PS-DVB-C18 particles DNASep column in which the unique HPLC profile generated from each specific amplicon represented corresponding Vibrio species. Our optimized mPCR-HPLC assay showed a higher sensitivity (ca. 101 CFU/mL (g) pure culture or food homogenate) than that of gel electrophoresis technique. Specificity of the mPCR-HPLC assay was tested on a total of 227 strains of Vibrio species and 89 non-Vibrio species. Specific HPLC peak profiles were produced in strains belonging to the target Vibrio species, but not in all strains other than the five species, showing high specificity of the assay. A total of 7255 seafood samples were collected to evaluate diagnostic capability of the mPCR-HPLC assay and results showed that 339 samples were positive in this assay, in accordance with the testing results of conventional culture-, biochemical-based assays. Results indicated that the mPCR-HPLC assay developed in this study was applicable for simultaneous detection of the five pathogenic Vibrio species from seafood in routine laboratories, providing a useful supplement to conventional methods for routine monitoring and risk assessment of seafood.

N-Acetyl-S-(N,N-diethylcarbamoyl) cysteine in rat nucleus accumbens, medial prefrontal cortex, and in rat and human plasma after disulfiram administration

Disulfiram (DSF), a treatment for alcohol use disorders, has shown some clinical effectiveness in treating addiction to cocaine, nicotine, and pathological gambling. The mechanism of action of DSF for treating these addictions is unclear but it is unlikely to involve the inhibition of liver aldehyde dehydrogenase (ALDH2). DSF is a pro-drug and forms a number of metabolites, one of which is N-acetyl-S-(N,N-diethylcarbamoyl) cysteine (DETC-NAC). Here we describe a LCMS/MS method on a QQQ type instrument to quantify DETC-NAC in plasma and intracellular fluid from mammalian brain. An internal standard, the N,N-di-isopropylcarbamoyl homolog (MIM: 291>128) is easily separable from DETC-NAC (MIM: 263>100) on C18 RP media with a methanol gradient. The method's linear range is 0.5–500nM from plasma and dialysate salt solution with all precisions better than 10% RSD. DETC-NAC and internal standards were recovered at better than 95% from all matrices, perchloric acid precipitation (plasma) or formic acid addition (salt) and is stable in plasma or salt at low pH for up to 24h. Stability is observed through three freeze-thaw cycles per day for 7 days. No HPLC peak area matrix effect was greater than 10%. A human plasma sample from a prior analysis for S-(N,N-diethylcarbamoyl) glutathione (CARB) was found to have DETC NAC as well. In other human plasma samples from 62.5mg/d and 250mg/d dosing, CARB concentration peaks at 0.3 and 4nM at 3h followed by DETC-NAC peaks of 11 and 70nM 2h later. Employing microdialysis sampling, DETC-NAC levels in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and plasma of rats treated with DSF reached 1.1, 2.5 and 80nM at 6h. The correlation between the appearance and long duration of DETC-NAC concentration in rat brain and the persistence of DSF-induced changes in neurotransmitters observed by Faiman et al. (Neuropharmacology, 2013, 75C, 95–105) is discussed.

In Vivo Evaluation of the Antiasthmatic, Antitussive, and Expectorant Activities and Chemical Components of Three Elaeagnus Leaves.

The leaf of Elaeagnus lanceolata and Elaeagnus henryi as well as Elaeagnus pungens has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional clinical medicine. This study was aimed at evaluating the antiasthmatic, antitussive, and expectorant activities of the water extracts from the three plants in vivo and analyzing their chemical components by HPLC-DAD. At the medium and high doses, the water extracts of three Elaeagnus leaves significantly prolonged the preconvulsive time (P < 0.01) in guinea pigs, lengthened the latent period of cough (P < 0.01) and decreased the cough frequency caused by aqueous ammonia in mice (P < 0.01), and enhanced tracheal phenol red output in mice (P < 0.01). There were no significant differences in the pharmacological actions between the three Elaeagnus leaves. Moreover, there was more similarity on overlap peaks in the range of retention time from 10 to 40 min by HPLC and many peaks that belonged to flavonoids compounds. It suggested that the main constituents of the three Elaeagnus leaves were flavonoid for the pharmacological activities. These effects were the important evidence for the traditional use of E. henryi leaf and E. lanceolata leaf as well as E. pungens to treat asthma and chronic bronchitis.

Alkaloids from Solanum paniculatum L. leaves

The roots, stems, leaves and fruits of Solanum paniculatum L. are used in Brazilian folk medicine as well as in culinary [1]. This species was formerly included in Brazilian Pharmacopoeia, but was removed from newer editions, due to the lack of scientific evidence on its medicinal use. Aiming at the expansion of the number of herbal medicines available for basic pharmaceutical care S. paniculatum L. was included by Brazilian Ministry of Health in a list of herbal medicines to guide research on their efficacy and safety. Solanum species are known to synthesize steroidal glycoalkaloids (SGA) in its leaves, roots and fruits. Jurubin, a 3β-amino-5α-furostane alkaloid was isolated from S. paniculatum L. [2]. The present report focus on the results of the phytochemical analysis an alkaloid fraction, obtained from leaves of S. paniculatum L.. Acid hydrolysis of a sample of the alkaloid enriched fraction produced five genins (ESI/HRMS analysis, 2 x C27H44NO2, C27H46NO2, C27H44NO3, C27H46NO3) and only one sugar, identified as glucose (GC/FID of the alditol acetate). These results confirmed that all the alkaloids had genins of the furostane type and were all linked to glucose. HPLC/ESI/HRMS of the alkaloid enriched fraction showed five peaks with formulae: A, C33H58NO8; C33H56NO8 (two peaks, B and C); D, C33H57NO9 and E, C34H59NO8. The spectra of these compounds displayed fragment masses corresponding to the elimination of one hexose plus one water (180 mass units) confirming the results found in the hydrolysis assay. Thus, the data for A, the main peak in HPLC is consistent with O(26)-β-D-glucopyranosyl 3β-amino-5α-furostane-22α,26-diol (jurubin) and the remaining formulae are in accordance with glycosylated 3β-amino-5α-furostanes structures with different degrees of oxygenation and unsaturation.

Synchronous Fluorescence Spectroscopy for Rapid Classification of Fruit Spirits

Fluorescence spectroscopy provides rapid profiling of food products and could become an effective tool for authentication when coupled to chemometrics. This study developed a simple method for classifying commercial fruit (apple, apricot, pear, and plum) spirits using synchronous fluorescence spectroscopy. Spectra were collected in the excitation wavelength range from 200 to 500 nm, with constant wavelength differences from 10 to 100 nm, and those obtained at wavelength differences 10, 90, and 100 nm were employed in multivariate analysis. Of samples, 100, 90, and 90 % were properly classified by applying the linear discriminate analysis to the first principal components of the principal component analysis performed on the synchronous fluorescence spectra at wavelength differences 10, 90, and 100 nm, respectively. One hundred percent of samples were properly classified by applying the general discriminate analysis to spectra regardless of wavelength difference used. For the comparison, HPLC analysis was also carried out and discrimination models were generated. The best results were obtained with the general discriminate analysis applied directly to the peak area of common HPLC peaks (correct classification 100 %, training set; 95 %, prediction set).

Synthesis and characterization of an antibacterial hydrogel containing covalently bound vancomycin.

The release of freely loaded small molecules from biomaterials often exhibits an initial burst, inhibiting the ability of these materials to match drug release with the biomaterial's degradation period. In terms of antibiotic release systems, the remaining vehicle may become a substrate for colonization by bacterial biofilms once the payload is depleted, which can become life threatening. Secondary surgeries are typically performed to remove these empty depots as a means of preventing this type of infection. To maintain the effectiveness of a locally delivered antibiotic without the drawback of a second surgery, we propose a hydrogel drug delivery system in which the drug release rate of vancomycin and degradation rate of the hydrogel are linked via covalent incorporation of vancomycin in the hydrogel backbone. This was achieved through coupling PEG based monomer with vancomycin to create poly(β-amino ester) chemistry and verified through drug release and matrix degradation studies. Antibiotic release and material degradation were tunable via hydrophobic/hydrophilic content of the hydrogel matrix and extended up to 3 weeks in PBS sink conditions. Covalent addition of vancomycin to the hydrogel polymer backbone was verified through mass spectroscopy and HPLC peak addition, as well as radiotracing of collected HPLC fractions. Bioactivity of released vancomycin was also confirmed alongside the resulting antimicrobial activity of the reacted vancomycin releasate.

Production, HPLC analysis, and in situ apoptotic activities of swainsonine toward lepidopteran, Sf-21 cell line.

Swainsonine, a secondary metabolite from Metarhizium anisopliae has been extensively studied in the complementary areas of therapeutics and toxicology. This work aims to develop a simple UV-HPLC method of analyses for swainsonine in Metarhizium fermentation broth and to explore its in situ entomotoxic activities. The partially purified broth was quantitatively analyzed using middle UV (205 nm)-reverse phase HPLC method with different mobile phases and gradient programmes. Swainsonine was eluted as single peak at (te ) 6.0-6.9 min with average concentration of 4.04 ± 0.52 μg/mL using optimal mobile phase (0.1% trifluoroacetic acid in water and acetonitrile). The mass spectrometry analysis further indicated the characteristic MS1 species for swainsonine, [M+H](+) 174.30 in corresponding HPLC peaks. The antiproliferative effects of swainsonine on lepidopteran, Sf-21 cells were determined through 3-(4, 5-dimethylthia-zol-2-yl)-2, 5-diphenyl tetrazolium bromide (IC50 standard = 3.90 μM and IC50 purified = 5.27 μM) and trypan blue dye exclusion (IC50 standard = 6.91 μM and IC50 purified = 8.67 μM) assays. The fluorescence activated cell sorting evaluation of Sf-21 cells showed nearly 35% and 42% of population in various apoptotic stages at 36 h, when treated with standard and purified swainsonine, respectively. The morphodimensional field emission scanning electron and atomic force microscopic analyses further confirmed the characteristic apoptotic features like membrane blebbings, ruptures and volume shrinkage in the lepidopteran cells after 24-36 h of post-treatment incubation. The study describes the potential entomotoxic activities of swainsonine and its role in the virulence of Metarhizium spp.

Analysis of benzo[c]phenanthridine alkaloids inEschscholtzia californicacell culture using HPLC-DAD and HPLC-ESI-MS/MS

Effective HPLC-DAD and HPLC-ESI-MS/MS methods have been developed for the analysis of eight benzo[c]phenanthridine alkaloids (sanguinarine, chelirubine, macarpine, chelerythrine, dihydrosanguinarine, dihydrochelirubine, dihydromacarpine and dihydrochelerythrine), which are important metabolites in Eschscholtzia californica cell culture. By adopting a ternary gradient pump system, the dihydro-form alkaloids hardly separable from each other could be successfully separated, and all the target alkaloids could be simultaneously quantified with the LOD values of 0.01-0.79 μg/mL and the LOQ values of 0.03-3.59 μg/mL. This HPLC-DAD method was further confirmed by HPLC-ESI-MS/MS system in multiple reaction monitoring mode. Each separated HPLC peak was identified as the target alkaloid, showing its relevant ionized molecule and selected fragment ion. By applying the established method, alkaloid production during the E. californica cell culture could be successfully monitored and some valuable information on its metabolism could be deduced.

Combining HPLC–DAD and ICP-MS data for improved analysis of complex samples: Classification of the root samples from Cortex moutan

A combined data matrix consisting of high performance liquid chromatography–diode array detector (HPLC–DAD) and inductively coupled plasma-mass spectrometry (ICP-MS) measurements of samples from the plant roots of the Cortex moutan (CM), produced much better classification and prediction results in comparison with those obtained from either of the individual data sets. The HPLC peaks (organic components) of the CM samples, and the ICP-MS measurements (trace metal elements) were investigated with the use of principal component analysis (PCA) and the linear discriminant analysis (LDA) methods of data analysis; essentially, qualitative results suggested that discrimination of the CM samples from three different provinces was possible with the combined matrix producing best results. Another three methods, K-nearest neighbor (KNN), back-propagation artificial neural network (BP-ANN) and least squares support vector machines (LS-SVM) were applied for the classification and prediction of the samples. Again, the combined data matrix analyzed by the KNN method produced best results (100% correct; prediction set data). Additionally, multiple linear regression (MLR) was utilized to explore any relationship between the organic constituents and the metal elements of the CM samples; the extracted linear regression equations showed that the essential metals as well as some metallic pollutants were related to the organic compounds on the basis of their concentrations.

Classification of Juniper-Flavoured Spirit Drinks by Multivariate Analysis of Spectroscopic and Chromatographic Data

This study compares the use of UV absorption, excitation–emission matrix (EEM) fluorescence and synchronous fluorescence spectroscopy and HPLC with fluorescence detection combined with principal component analysis (PCA), parallel factor analysis (PARAFAC) and linear discriminant analysis (LDA) for distinguishing between commercial samples of Slovak, Belgian, German, Czech and British juniper-flavoured spirit drinks. Overall, 97 %, 88 % and 79 % of samples were properly classified by applying the LDA to the first five principal components of the PCA performed on the synchronous fluorescence spectra (constant wavelength difference 10 nm, 250–450 nm), UV absorption spectra (250–325 nm) and the areas of eight common HPLC peaks, respectively. EEM fluorescence spectroscopy could not discriminate British drinks, while HPLC failed to discriminate Belgian samples. When the areas of eight common HPLC peaks were used as model parameters instead of the five PCs initially used in LDA, the method accuracy was enhanced significantly and 97 % of predicting samples were properly classified.

Comparison of extraction methods for the analysis of Indigofera tinctoria and Carthamus tinctorius in textiles by high performance liquid chromatography

The extractions of indigo (Indigofera tinctoria L.) and safflower (Carthamus tinctorius L.) red and yellow from wool fibres are investigated using high performance liquid chromatography with photo-diode-array detection (HPLC–PDA) which is frequently coupled to Mass Spectrometry (HPLC–PDA–MS) for identification purposes. The efficiencies of dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) to extract indigo and safflower from wool, are compared for a wide range of temperature (from 40 to 120°C) and treatment time (from 1 to 30min). Extraction procedures are investigated by measuring integrated HPLC peak areas of selected dyestuff components, evaluating simultaneously the effects of solvent (DMSO and DMF), treatment temperature (T) and time (t). Thus, cross-influence of the different extraction parameters (solvent, T and t) is taken into account. Indigotin, isatin and indirubin are monitored to evaluate the extraction of indigo. Carthamin, a decomposition product of carthamin, apigenin, safflomin A, 6-hydroxykaempferol-3-O-β-D-glucoside, anhydrosafflor yellow Ct1 and Ct4 are monitored to investigate the extractions of safflower red and yellow. Overall, it is reported that DMSO gives better extraction yields than DMF.The best conditions for the extraction of the marker compounds of the three dyes are summarized as follows. For indigotin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for t=1min give the best yield. Longer treatment (t>1min) at 120°C results to decomposition of indigotin. For carthamin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for short t (<5min) give the best yield, considering that longer (>5min) treatment at 120°C results to decomposition of the marker compound. Finally, for safflomin A treatments of the wool with DMSO at 100°C for t>10min or at 120°C min for t=5min give the best result.

Estrogen receptor affinity chromatography: A new method for characterization of novel estrogenic disinfection by-products

To identify the unknown estrogenic disinfection by-products (DBPs) from the chlorination extract, an effective method based on affinity chromatography with immobilized human recombinant estrogen receptor α (ERα) was developed, which has an advantage in targeting different potential estrogenic compounds from mixed sample simultaneously by comparing their relative binding activities to ER. The new method worked well for six known environmental estrogens. To further test the validity of this method for unknown chemicals, six DBPs of diethylstilbestrol (DES) with relatively strong ER binding affinity after chlorination were isolated and identified. It was found that except for 2-chloro-DES which showed 1.36 times stronger binding affinity than DES, most of the by-products bound to ER much more weakly than DES. All these seven by-products induced a dose-dependent transcriptional activation in two-hybrid-yeast assays. Z,Z-dienestrol (DE) and 2-chloro-DES, which exhibiting the weakest and the strongest binding affinity, were further tested for their transcriptional potential as 0.00243 and 0.014 compared to DES, respectively. However, they were still potential harmful environmental estrogenic disruptors as their estrogenic activities were much stronger than that of bisphenol A (BPA). These results demonstrated that the new method can help to screen unknown estrogenic compounds from mixture more efficiently.

Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in Fusarium verticillioides

Fumonisins are agriculturally important mycotoxins produced by the maize pathogen Fusarium verticillioides. The chemical structure of fumonisins contains two tricarballylic esters, which are rare structural moieties and important for toxicity. The mechanism for the tricarballylic ester formation is not well understood. FUM7 gene of F. verticillioides was predicted to encode a dehydrogenase/reductase, and when it was deleted, the mutant produced tetradehydro fumonisins (DH4–FB). MS and NMR analysis of DH4–FB1 indicated that the esters consist of aconitate with a 3′-alkene function, rather than a 2′-alkene function. Interestingly, the purified DH4–FB1 eventually yielded three chromatographic peaks in HPLC. However, MS revealed that the metabolites of the three peaks all had the same mass as the initial single-peak DH4–FB1. The results suggest that DH4–FB1 can undergo spontaneous isomerization, probably including both cis–trans stereoisomerization and 3′- to 2′-ene regioisomerization. In addition, when FUM7 was expressed in Escherichia coli and the resulting enzyme, Fum7p, was incubated with DH4–FB, no fumonisin with typical tricarballylic esters was formed. Instead, new fumonisin analogs that probably contained isocitrate and/or oxalosuccinate esters were formed, which reveals new insight into fumonisin biosynthesis. Together, the data provided both genetic and biochemical evidence for the mechanism of tricarballylic ester formation in fumonisin biosynthesis.

Sequential elution liquid chromatography can significantly increase the probability of a successful separation by simultaneously increasing the peak capacity and reducing the separation disorder

This paper demonstrates that sequential elution liquid chromatography (SE-LC), an approach in which two or more elution modes are employed in series for the separation of two or more groups of compounds, can be used to separate not only weak acids (or weak bases) from neutral compounds, but weak acids and weak bases from neutral compounds (and each other) by the sequential application of either of two types of an extended pH gradient prior to a solvent gradient. It also details a comparison, based on peak capacity and separation disorder, of the probability of success of this approach with the unimodal elution approach taken by conventional column liquid chromatography. For an HPLC peak capacity of 120 and samples of moderate complexity (e.g., 12 components), the probability of success (Rs≥1) increases from 37.9% (HPLC) to 85.8% (SE-LC). Different columns were evaluated for their utility for SE-LC using the following criteria: (1) the prediction of the elution order of the groups based on the degree of ionization of the compounds; and (2) the closeness of the peak shape to the ideal Gaussian distribution. The best columns overall were the Zorbax SB-AQ and Waters XBridge Shield columns, as they provided both between-class and within-class separations of all compounds, as well as the lowest degree of tailing of 4-ethylaniline using the pH 2 to pH 8 gradient.

Identification of putative quantitative trait loci associated with a flavonoid related to resistance to cabbage seedpod weevil (Ceutorhynchus obstrictus) in canola derived from an intergeneric cross, Sinapis alba × Brassica napus.

Kaempferol 3- O -sinapoyl-sophoroside 7- O -glucoside was putatively identified as the major component of a characteristic HPLC peak previously correlated with the reduction of cabbage seedpod weevil larval infestation in a novel canola genotype. The cabbage seedpod weevil (Ceutorhynchus obstrictus [Marsham]) (CSPW) is a serious pest of brassicaceous oilseed crops such as canola in both Europe and more recently in North America. At present, the only control strategy against CSPW is the application of insecticides. As an alternative more environmentally-friendly control strategy, we developed novel canola germplasm resistant to weevil attack through introgression of Sinapis alba DNA into Brassica napus by making the wide cross followed by embryo rescue and backcrossing to the B. napus parent. We have previously characterized resistant canola lines by metabolic profiling and were able to correlate reduction of larval infestation to the presence of a characteristic HPLC peak. In this study, we have putatively identified the major component in the peak using mass spectrometry as kaempferol 3-O-sinapoyl-sophoroside 7-O-glucoside (KSSG). We have also identified quantitative trait loci (QTL) associated with this HPLC peak in a mapping population consisting of more than 200 individual doubled haploid (DH) lines derived from a cross between CSW428 (the resistant parent) and SC030686 (the susceptible parent). This QTL accounted for approximately 9.5% of the phenotypic variation in KSSG content. The observation that only one QTL was identified as surpassing the LOD threshold of 3.0 suggests that both parents may possess the positive alleles for other QTL that have not been detected in our study. This finding also indicates a complex regulatory mechanism for KSSG levels and provides an appropriate explanation for the large transgressive segregation observed in the DH lines of the QTL mapping population.

Comparative Evaluation of Antimutagenicity of Commonly Consumed Fruits and Activity-Guided Identification of Bioactive Principles from the Most Potent Fruit, Java Plum (Syzygium cumini)

Commonly consumed fruits showed remarkable variation in antimutagenicity when assayed by E. coli rifampicin resistance assay, and Java plum (Syzygium cumini) was found to be one of the most potent fruits. Its anthocyanins contributed maximally to the observed antimutagenicity and resolved into three distinct bands in HPTLC. Although these bands displayed similar antioxidant capacity, the band at R(f) 0.22 was the most antimutagenic and resolved into two peaks in HPLC. The second peak (t(R) 3.8 min) displayed a strong and broad spectrum antimutagenicity and was identified as petunidin-3,5-diglucoside by analysis of its molecular ion and fragmentation pattern by ESI-MS/MS. The presence of glucose moiety was confirmed by TLC analysis of acid-hydrolyzed products. This purified anthocyanin was found to suppress mutagenic SOS DNA repair process in E. coli and thus indicated suppression of the error-prone DNA repair pathway as one of the major mechanisms of antimutagenicity of this fruit.

Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2%), bi-(23.9%), tri-(9.0%), tetra-(2.7%), and penta-(2.8%) antennary oligosaccharides. However, OM contained mostly tri-(33.5%) and penta-(31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21-22°C), the rest remaining in the liquid state in the 5°C-45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.

Effects of soaking, boiling and autoclaving on the phenolic contents and antioxidant activities of faba beans (Vicia faba L.) differing in seed coat colours

The Australian grown faba beans of different seed coat colours were either soaked, boiled or autoclaved, and analysed for phenolic contents and antioxidant activity using an array of reagent-based assays. Soaking, boiling and autoclaving were shown to lower the level of active compounds in faba beans. A significant amount of active compounds was leached to the soaking and cooking medium. Boiling was a better method in retaining active compounds in beans than autoclaving. The boiled beans had more active compounds than those of resulting cooking broths, which was the opposite observation when autoclaving. The buff-genotypes had a similar level of active compounds to red- and green-genotypes. The high performance liquid chromatography-post column derivatisation (HPLC-PCD) system detected a dense collection of high antioxidant HPLC peaks ('humps') in extracts of raw, soaked and boiled beans. The present findings encouraged consumption of faba beans together with cooking broth for the maximum potential health benefits.