O-phthalaldehyde Research Articles

Selective extraction of thiol-containing peptides in seawater using Tween 20-capped gold nanoparticles followed by capillary electrophoresis with laser-induced fluorescence

This study combines Tween 20-capped gold nanoparticles (Tween 20-AuNPs) with capillary electrophoresis (CE) for ultrasensitive detection of thiol-containing peptides, including glutathione (GSH), γ-glutamylcysteine (γ-GCS), and phytochelatin analogs. By forming AuS bonds, Tween 20-AuNPs can selectively extract and enrich these thiols from a complicated matrix. A Tween 20 capping layer not only suppresses nonspecific adsorption, but also enables NPs to disperse in a highly salinity solution. Dithiothreitol removes thiol-containing peptides from the NP surface through ligand exchange. The released peptides are selectively derivatized with o-phthaldialdehyde (OPA) to form tricyclic isoindole derivatives. Extraction efficiency of five thiol-containing peptides with Tween 20-AuNPs was highly reliable in the Tween 20-AuNP concentration, time of extraction and desorption thiols, and sample volume. After injecting a large sample volume, the OPA-derivatized peptides migrate against the electroosmotic flow (EOF) and enter the polyethylene oxide (PEO) zone. The sensitivity of these peptides was improved by stacking them at the boundary between the sample and PEO zones. As a result, limits of detection (LODs) for five peptides were down to 0.1-6 pM. Not only is the proposed method probably the first CE example for detecting dissolved thiols in seawater; it also has the lowest LODs for GSH, γ-GCS, and phytochelatins compared to other reported methods.

Development of a fluorescence-based microplate method for the determination of volatile fatty acids in anaerobically digested and sewage sludges

This paper presents a simple, accurate and multi-sample method for the determination of volatile fatty acids (VFAs) thanks to a 96-well microplate technique. A procedure using an activating reagent of the carboxylic function (water-soluble carbodiimide EDC) and a fluorescent amino labeling reagent (N-(1-naphthyl)ethylenediamine, EDAN) allows the formation of an isoindole derivative that needs to be separated from initial fluorescent amine for efficient VFAs determination. Isolation of these fluorescent VFA-derivatives was carried out by use of the fluorescent quenching of EDAN with o-phthaldialdehyde (OPA). Quenching was most efficient at pH around 7 and by heating at 40°C within the microplate reader. This optimized procedure has been applied to various carboxylic acids and other organic compounds, demonstrating that VFA exhibit the highest fluorescence responses with homogeneous results for the main ones (acetic, propionic and butyric acid, all mass concentration expressed as acetic acid equivalents). This protocol was calibrated against acetic acid and determination of VFA was thus possible in the range 3.9–2000mgL−1 (acetic acid equivalents). Subsequent application to real samples (sewage sludges or anaerobically digested samples) and comparison to gas chromatography analyses gave accurate results, proving the great potential of our high-throughput microplate-based technique for the analysis of VFA.

Comparative antioxidant activity, proteolysis and in vitro α-amylase and α-glucosidase inhibition of Allium sativum-yogurts made from cow and camel milk

The presence of extract of plant with medicinal properties during milk fermentation could enhance the therapeutical values of yogurt. In the present study, the effects of Allium sativum on the changes in post-acidification, total phenolic content (TPC), proteolysis by o-phthaldialdehyde (OPA) assay, antioxidant activity by (1,1-diphenyl-2-picrylhydrazyl radical (DPPH) inhibition) and capacity to inhibit in vitro α-amylase and α-glucosidase activities in cow or camel milk yogurt (MY) during 21day refrigerated storage were investigated. The presence of A. sativum enhanced more pH reduction for camel-MY than for cow-MY compared to their respective controls during storage. The reverse was true for total titratable acid. TPC in camel-MY was higher (p<0.05) than that in cow-MY. The presence of A. sativum in cow- and camel-MYs elevated (p<0.05) the TPC, but these changed little during storage. Antioxidant activities (18–38% DPPH inhibition) were not different in both types of yogurts, either in the absence or in the presence of A. sativum. However, camel-MY had an increase (p<0.05) in antioxidant activities (49–65%) during 7–21days of storage. OPA values on day 0 was higher for camel-MY (368.2±14.8mg/g) than for cow-MY (80.1±3.2mg/g). The presence of A. sativum increased OPA values more for cow-MY than for camel-MY (3.0- and 1.3-folds, respectively). Higher inhibition (p<0.05) of α-amylase by camel-MY compared to cow-MY occurred whereas α-glucosidase inhibition by cow-MY reduced (p<0.05) as a result of refrigeration greater than 7days. In general, the addition of A. sativum caused more antioxidant activities, proteolysis and enzymes (α-amylase and α-glucosidase) inhibition in camel-MY than in cow-MY.

ChemInform Abstract: N‐Heterocyclic Carbene Catalyzed Intramolecular Crossed Aldehyde—Ketone Benzoin Condensation in the Chalcone of o‐Phthalaldehyde: A Facile Synthesis of Naphthalenones.

Abstract The intramolecular crossed aldehyde–ketone benzoin condensation in the chalcone of o -phthalaldehyde (OPA) catalyzed by N -heterocyclic carbene (NHCs) generated in situ from readily available imidazolium and thiazolium salts is described. In this reaction, bicyclic α-hydroxyl ketones (naphthalenone type tertiary alcohol) were selectively produced in good yields (75–94%) in shorter reaction times (20 min) through nucleophilic addition of acyl anion generated by umpolung in OPA–chalcone (regio controlled).

A FLUORESCENCE-LC METHOD WITH NDA PRE-COLUMN DERIVATIZATION FOR FUMONISIN B2 DETERMINATION IN BLACK ASPERGILLI CULTURES

The recent discovery that Aspergillus niger can produce fumonisins raises a new mycotoxigenic risk that requires additional studies to assess the impact on agricultural commodities. Here, we report the development and validation of a sensitive LC-fluorescence method for the identification of fumonisin B2 in black aspergilli cultures. The performances of naphthalene-2,3-dicarboxaldehyde (NDA), o-phthaldialdehyde (OPA), and 9-fluorenylmethyl chloroformate (FMOC) for the precolumn derivatization of samples were evaluated and compared. The best methodology included the following elements: NDA was used during pre-column derivatization; the derivatives were separated by a 30 min isocratic elution with acetonitrile/H2O/acetic acid (60:40:1, v/v/v), pumped at a flow rate of 1 mL · min−1 on a polar C18 reversed-phase YMC-Pack ODS-AQ column (250 × 4.6 mm, 5 µm); and fluorescence detection using excitation and emission wavelengths of 420 and 500 nm, respectively. The calibration curve was found to give a good linear response (r2 > 0.9999) from 1 to 200 µg · L−1. For FB2, which is the main fumonisin detected in black aspergilli thus far, this method measured an LOD and an LOQ of 2 and 6 µg · kg−1, respectively. Using this method, the amount of FB2 in black aspergilli cultures can be simply and rapidly determined.

Development of a simple capillary electrophoretic determination of glucosamine in nutritional supplements using in-capillary derivatisation with o-phthalaldehyde

A simple capillary electrophoretic method was developed for the determination of glucosamine using in-capillary derivatisation. Glucosamine in commercial products was extracted with purified water. The CE separation was achieved on an uncoated fused-silica capillary using a 20mM borate buffer (pH 9.2) containing 5mM o-phthalaldehyde (OPA) and 5mM 3-mercaptopropionic acid (MPA) at 25kV, followed by UV detection at 340nm. The detector response was linear (r2>0.999) in the concentration range 10–1000μg/mL. The limit of detection (LOD) was 1.3mg/g. Spiked glucosamine recoveries at 50 and 100mg/g level were 95.1% and 104.3%, respectively. The method was applied to 16 commercial products. The concentrations of glucosamine were 109–705mg/g, and the ratios of detected glucosamine content to the labelled value were 88.8–124%. No significant bias was observed (r2=0.989, p<0.01), between results obtained by the proposed CE method and an official colorimetric method (Japanese Health Food & Nutrition Food Association).

Differentiation of Allium subgenera by cysteine sulphoxides and further amino acids

The genus Allium (>800 species) has been divided into several subgenera, sections, species and subspecies [1]. Cysteine sulphoxides are most prominent amino acid derivatives of this genus. These substances are susceptible for the enzyme alliinase. Alllinase reaction and further non-enzymatic steps do result in volatile sulphur compounds, which exhibit various bioactivities, e.g., antibiotic activities. The pattern of cysteine sulphoxides is rather characteristic for some subgenera. For instance, the subgenus Melanocrommyum is characterized by different heteroaromatic cysteine sulphoxides, whereas the subgenus Cepa typically shows rather high amounts of isoalliin. In the now presented study, amino acids as well as cysteine sulphoxides have been analyzed as their corresponding o-phthaldialdehyde (OPA) derivatives. Besides the above mentioned differences in the pattern of cysteine sulphoxides, also further amino acids show some relations between their amounts and subgenera. Most prominent are differences in the content of L-arginine in different subgenera of the genus Allium. L-arginine is a valuable nitrogen storage compound in many angiosperms [2]. But this amino acid has been also found in some Allium species in high concentrations. As an example, the amount of L-arginine in Allium cepa L. was increased during the maturation process [3]. Further on, the amount of L-arginine in samples belonging to the subgenera Allium and Cepa was significant higher (p<0.05) as the amount of L-arginine found in the subgenus Melanocrommyum suggesting different mechanisms of nitrogen storage. These differences should allow chemo-taxonomical classification of subgenera.

Allium Species from Central and Southwest Asia Are Rich Sources of Marasmin

Marasmin, which is especially known from the two South African species Tulbaghia alliacea and Tulbaghia violacea , but was also described for the garlic mushroom Marasmius alliaceus , is the precursor of the thiosulfinate marasmicin. Marasmicin has attracted considerable attention because of its antifungal and tuberculostatic activities. However, many Allium species of the subgenus Melanocrommyum, especially Allium suworowii , are also very rich in marasmin. A. suworowii revealed concentrations of marasmin up to 1.6%, related to the fresh weight of bulbs, and up to 3.0%, related to air-dried fruiting bodies, of the corresponding γ-glutamylmarsmin was found in M. alliaceus. Both species show much higher amounts of marasmin as Tulbaghia and could be considered as natural sources for the isolation of this compound. Further promising Allium species with considerable amounts of marasmin besides other cysteine sulfoxides are Allium stipitatum and Allium altissimum . (R(S),R(C))-Marasmin is typical for the investigated species of the subgenus Melanocrommyum, whereas γ-glutamyl-(S(S),R(C))-marasmin is the only cysteine sulfoxide for the genus Marasmius known until now. Both cysteine sulfoxides were isolated and described as o-phthaldialdehyde (OPA) derivatives. Furthermore, the cysteine sulfoxides methiin, propiin, S-(2-pyrrolyl)-cysteine sulfoxide, eventually S-(2-pyridyl)-cysteine sulfoxide and S-(2-pyridyl)-L-cysteine N-oxide were found.

Separation optimization in reversed-phase liquid chromatography by using alkanol additives in the mobile phase: Application to amino acids

In an effort to enhance complex mixture separations by using small amounts of a homologous series of alkanols as additives in the mobile phases, it was proposed an optimization algorithm based on a sixth-parameter retention model. This model considers simultaneously the contents of the main organic modifier and of the alkanol additive in the mobile phase as well as of the number of alkyl chain of the additive. This model is in fact a modification of a previously one derived in a recently published paper for the retention description of a mixture of purely hydrophobic alkylbenzenes under isocratic conditions with mobile phases containing alkanol additives. The effectiveness of the new retention model as well as the optimization algorithm was successfully applied to the separation of ten o-phthalaldehyde (OPA) derivatives of amino acids. Indeed, the new retention model exhibited an excellent prediction performance since the obtained overall predictive error between calculated and experimental times was only 2.8% for all isocratic runs by using a variety of mobile phase compositions containing any alkanol homologue even different than those used in the starting/fitting experiments. Moreover, a perfect resolution of the above amino acid mixture was achieved within only 7.4min in the chromatogram recorded using the optimal mobile phase determined by means of the simple optimization algorithm proposed in this study.

The effect of mild Pulsed Electric Field (PEF) conditions on acid tolerance, bile tolerance, growth and protease activity of the Lactobacillus delbrueckii ssp. bulgaricus LB-12.

Pulsed electric field (PEF) processing involves the application of pulses of voltage for less than one second to fluid products placed between two electrodes. So this study was conducted for the quantitative assessments of mild PEF conditions on acid tolerance, growth, bile tolerance and protease activity of Lactobacillus delbrueckii ssp. bulgaricus LB-12. The control and PEF treated samples were prepared by inoculating 10 ml of freshly thawed culture of Lactobacillus delbrueckii ssp. bulgaricus LB-12 into 990 ml of sterile 0.1% (wt/v) peptone water and treated in a pilot plant PEF system (OSU-4M). The treatments were positive square unipolar pulse width of 3 μs, pulse period of 0.5 second, voltage of 1 kV/cm, delay time of 20 μs and flow rate of 60 ml/min at 40.5°C. The control was passed through the PEF system (60 ml/min) without receiving any pulsed electric field condition. The acid tolerance was determined every 30 minutes for 120 minutes of incubation in acidified MRS broth at pH 2. Growth was determined hourly for 25 hours of incubation at 37°C in MRS broth. The bile tolerance was determined hourly for 16 hours of incubation in MRS-Thio broth . Samples were plated in duplicates using pH modified Lactobacilli MRS agar. The petriplates were incubated anaerobically at 43C for 72 hours. Protease activity was determined by o-phthaldialdehyde (OPA) UV- spectrophotometric assay at 0, 12, 24, 36 and 48 hours of incubation of conducted. inoculated skim milk at 40°C. Three replications were The control. The stationary phase of the bacterium was between hours 10 and 18. Moreover significant experimental design was repeated measurements on complete randomized block, Replications were the blocks. Data were analyzed using Proc Mixed model of Statistical Analysis System (SAS).The viability of the control Lactobacillus delbrueckii ssp. bulgaricus LB-12 was lost after 30 minutes of incubation in acidified MRS broth (pH 2), whereas, the bacterium subjected to mild PEF treatment was acid tolerant until the end of 120 minutes of incubation. Mild PEF significantly improved acid tolerance of Lactobacillus delbrueckii ssp. bulgaricus LB-12. and the growth reached the logarithmic phase an hour earlier than the decrease

HPLC determination of fumonisin mycotoxins in maize: A comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthaldialdehyde derivatization reagents for fluorescence and diode array detection

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B 1 (FB 1), fumonisin B 2 (FB 2) and fumonisin B 3 (FB 3) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n = 6) for FB 1, FB 2 and FB 3 in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples ( n = 15) ranging in total fumonisin (TFB = FB 1 + FB 2 + FB 3) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB 1, FB 2 and FB 3 were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.

Determination of apramycin in oral soluble powder by a HPLC method using pre-column derivatization with o-phthalaldehyde and UV detection

A high-performance liquid chromatographic method employing pre-column derivatization with o-phthalaldehyde (OPA) and 2-mercaptoacetic acid was developed for the determination of apramycin, an aminoglycoside antibiotic used in veterinary medicine, in the oral soluble powder form. The chromatographic separation was done by ion-pair HPLC using a C18 reversed-phase column, Synergy Hydro (150 mm x 4.6 mm x 4 µm) and mobile phase composed of 0.005 mol/L sodium octanosulfonate in a mixture of acetonitrile: water: acetic acid (45:55:2) (v/v/v) with a flow rate of 1.0 mL/min; the UV detector was operated at 332 nm. The developed method was validated according to official compendia guidelines, having demonstrated robustness, selectivity and linearity for the concentration range of 0.02 to 0.05 mg/mL, precision (with RSD &lt; 2.0% both for intra and inter-day precision) accuracy (average recuperation of 99.33%) and detectivity (quantification and detection limits of 0.08 and 0.02 µg/mL, respectively). Three batches of commercial apramycin oral soluble powder were analyzed by both the proposed method and the official microbiological method, where all the results obtained were in the acceptable range (95% to 105% of labeled value of apramycin). Both methods were statistically compared by the t test, which yielded no significant differences (α = 0.05) thereby confirming the equivalence of the methods.

Development and validation of a pre-column reversed phase liquid chromatographic method with fluorescence detection for the determination of primary phenethylamines in dietary supplements and phytoextracts

A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts ( Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ em = 455 nm with λ ex = 340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ em = 310 nm with λ ex = 275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5–101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment.

N-Heterocyclic carbene catalyzed intramolecular crossed aldehyde–ketone benzoin condensation in the chalcone of o-phthalaldehyde: a facile synthesis of naphthalenones

The intramolecular crossed aldehyde–ketone benzoin condensation in the chalcone of o-phthalaldehyde (OPA) catalyzed by N-heterocyclic carbene (NHCs) generated in situ from readily available imidazolium and thiazolium salts is described. In this reaction, bicyclic α-hydroxyl ketones (naphthalenone type tertiary alcohol) were selectively produced in good yields (75–94%) in shorter reaction times (20 min) through nucleophilic addition of acyl anion generated by umpolung in OPA–chalcone (regio controlled).

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3mg of N/mL of inoculum) and incubated with 10mL of ruminal inoculum and 5mL of buffer; fermentations were stopped after 2h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t=0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (kd) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters Km (Michaelis constant) and kd (Vmax/Km), where Vmax=maximum velocity, were estimated directly; kd values were adjusted (Akd) for the fraction FD0 using the equation Akd=kd − FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean=0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides.

A microchip electrophoresis strategy with online labeling and chemiluminescence detection for simultaneous quantification of thiol drugs

An integrated microfluidic device with online labeling and chemiluminescence (CL) detection was developed for the simultaneous quantification of thiol drugs. In this device, the online labeling, electrophoresis separation and CL detection were compactly integrated onto a glass/poly(dimethylsiloxane) (PDMS) hybrid microfluidic chip. CL detection was based on the oxidation reaction of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and o-phthalaldehyde (OPA) labeled thiol drugs with NaBrO. Four thiol drugs including 2-mercaptopropionylglycine (2-MPG), captopril (CP), 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP) were employed as model compounds to examine the utility of the system. It was indicated that the separation and detection of four drugs can be completed within 90 s. Detection limits (S/N = 3) for the thiol drugs tested were in the range of 8.9 × 10 −9–13.5 × 10 −9 M. The application of the present system was demonstrated by analyzing the thiol drugs in human plasma samples.

Determination of proline enantiomers in honey and royal jelly by LC-UV.

The chiral separation and quantification of D-proline and L-proline in honey and royal jelly were examined by LC with UV detection. Most of the endogenous compounds existing in honey, such as sugars, were removed by using SPE cartridges containing C18 and strong cation-exchange sorbent. Other components, such as primary amino acids, were also removed by two-step derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-CI). The components that were derivatized with OPA were separated from proline with a C18 cartridge. Proline was then converted into an FMOC derivative that could be subsequently measured by LC-UV. Sufficient chiral separation of D-proline and L-proline was achieved with an LC chiral column made of a beta-cyclodextrin phase in the polar organic-phase mode. The average recoveries of D-proline and L-proline from honey and royal jelly were in the range of 81.3-98.6% (RSD of < 1.8%). When this method was applied to commercial honey and royal jelly samples, L-proline was detected at concentrations of 369-1930 microg/g, whereas D-proline was not detected.

Changes in yogurt fermentation characteristics, and antioxidant potential and in vitro inhibition of angiotensin-1 converting enzyme upon the inclusion of peppermint, dill and basil

The present study investigated the effects of peppermint ( Mentha piperita), dill ( Anethum graveolens) and basil ( Ocimum basilicum) on yogurt formation, proteolysis and inhibition of angiotensin-1 converting enzyme (ACE). Herbal-yogurts had faster rates of pH reduction than plain-yogurt. All herbal-yogurts had higher ( p < 0.05) antioxidant activities than plain-yogurt, both at the end of fermentation and throughout the storage period. The o-phthalaldehyde (OPA) peptides in herbal-yogurts increased by 28–36% after 7 days of storage. All herbal-yogurts showed higher anti-ACE activity than plain-yogurt at corresponding storage periods. M. piperita yogurt had highest inhibitory effect on ACE activity throughout the storage period. Peppermint, dill and basil may be used to modify microbial fermentation of milk with the intention of producing dairy products with higher antioxidant and enhanced anti-ACE activities.

Purification and characterization of a 6.5 kDa antioxidant peptidoglycan purified from silk worm (Bombyx mori) pupae extract

A unique antioxidant peptidoglycan of 6.5 kDa was purified from silk worm (Bombyx mori) pupae. The peptidoglycan was extracted and isolated from the acidified aqueous extract of the homogenized pupae by chloroform/methanol (2:1, v/v), and purified by size-exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). This separation method allowed an easy and consistent preparation of the peptidoglycan from the high protein load by using only 3 steps of purification. The highly pure peptidoglycan consisted of fucose, glucosamine, galactosamine, glucose, and galactose as constituent sugars when revealed by high-performance anion exchange chromatography (HPAEC) analysis. The amino acid composition analysis showed the presence of acidic and polar amino acids (51.7%), hydrophobic (37.2%), and basic amino acids (9.3%) in the purified peptidoglycan. The IC50 of the purified peptidoglycan was 0.22±0.05 and 0.61±0.09 μg for diphenyl-2-picryl hydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively, as quantified by Ophthalaldehyde (OPA) microplate fluorescent assay.

Precolumn derivatization followed by liquid chromatographic separation and determination of tramiprosate in rat plasma by fluorescence detector: Application to pharmacokinetics

Alzheimer disease (AD) is characterized pathologically by extracellular amyloid deposits composed of amyloid β (Aβ) protein. A simple and rapid method using HPLC with fluorescence detector was developed and validated for determination of tramiprosate in rat plasma. Pre-column derivatization of the deproteinized rat plasma was carried out using o-phthaldialdehyde (OPA) as a fluorescent reagent in presence of 3-mercaptopropionic acid. The liquid chromatographic separation was achieved on a Kromasil C 18 column using methanol:acetonitrile: 20 mM phosphate buffer pH 7.5 (8.0:17.5:74.5 v/v/v) as a mobile phase in an isocratic elution mode. The eluents were monitored by a fluorescence detector set at 330 and 450 nm of excitation and emission wavelength respectively. Vigabatrin was used as an internal standard. The method was linear within the range 30.0–1000.0 ng/mL. Design of experiments (DOE) was used to evaluate the robustness of the method. The developed method was applied to study the pharmacokinetics of tramiprosate in rats.