Biomedical subjects

Irena Vovk

Publications and source records attributed to Irena Vovk.

12 recordsLinked to original sources

PubMed2006

Isolation of tomato pectin methylesterase and polygalacturonase on monolithic columns.

An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 microg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 microg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.

Carboxylic Ester Hydrolases↗

PubMed2006

Separation of pectin methylesterases and polygalacturonases on monolithic columns.

The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; M(r) 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with M(r) 82 kDa) and a basic protein with M(r) 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with M(r) 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH(3)CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.

Carboxylic Ester Hydrolases↗

PubMed2006

Analysis of sucrose esters--insecticides from the surface of tobacco plant leaves.

Sucrose esters from the surface of leaves of Nicotiana tabacum L. have been shown to possess interesting biological activities. We developed a simple and effective method for their analysis using HPTLC silica gel plates, n-hexane-ethyl acetate (1:3, v/v) as developing solvent and aniline-diphenylamine as a detection reagent. Off-line TLC-MS was also used for the detection and identification of the compounds. Solutions containing sucrose esters upon alkaline hydrolysis give sucrose, which is used for indirect estimation by TLC of the sucrose ester content. The method is applicable for the screening for sucrose esters in plant extracts. The extract obtained from the surface of green leaves of oriental tobacco type Prilep P-23 contains sucrose esters and is effective against Myzus persicae (Sulzer) in laboratory and field experiments.

Chromatography, Thin Layer↗

PubMed2006

The biological and chemical variability of yacon.

This paper focuses on the biological and chemical variability of four yacon (Smallanthus sonchifolius) accessions cultivated under field conditions. Significant variations in tuber shape, weight, content of oligofructans, as well as in leaf isozymes, phenolics, and relative DNA contents were found. Accessions 6 and 88 were the most productive (up to 3.01 and 3.74 kg/plant); accession 48 was the most balanced from the yield aspect in three vegetative periods. A significantly higher content of beta-(2-->1) oligofructans was noted in accessions 48 and 88 as compared to 6 and 60. No difference in sucrose, glucose, and fructose level was observed. Only accession 6 exhibited separate acid phosphatase and esterase isoforms. Accessions 6 and 60 had the highest content of phenolics, and accession 88 had the lowest relative DNA content. Large yacon intraspecific variation may be useful in future detailed research as a good background for breeding, growing, and utilization in industrial processing.

Asteraceae↗

PubMed2006

Intestinal permeability and cytokine inflammatory response in multiply injured patients.

In experimental settings, the increased intestinal permeability (IP) following severe trauma is associated with increased serum concentrations of cytokines. Multiply injured patients are susceptible to the development of multiple organ failure (MOF). The aim of this study was to determine if altered IP after trauma was associated with upregulation of cytokines and if cytokines and IP influenced the development of MOF. In 30 multiply injured patients, IP was measured on days 2 and 4 after injury using the lactulose-mannitol (L-M) test, and the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8 were determined simultaneously. The L-M ratio increased significantly from 0.049 (0.017-0.133) on day 2 to 0.150 (0.059-0.339) on day 4 (p < 0.02) On day 4, a significant correlation was also found between the L-M ratio and IL-6 (r = 0.43, p < 0.03). The IL-6 level on days 2 and 4 was significantly (p < 0.01 and p < 0.03, respectively) higher in MOF patients than in those without MOF, as was the TNF-alpha level on day 4 significantly higher (p < 0.04) in MOF patients. IP increases following multiple trauma, and on day 4 it correlates with the IL-6 level. However, in patients who develop MOF only cytokines are invariably increased, with IL-6 alone being significantly increased on both measurements in these patients.

Adult↗

PubMed2005

Separation of eight selected flavan-3-ols on cellulose thin-layer chromatographic plates.

The potential of microcristaline cellulose as sorbent in the separation of eight compounds: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), procyanidin B1 and procyanidin B2 was studied. Cellulose HPTLC plates prewashed in water (not necessary, when water was used as developing solvent) and dried with a hair dryer, bandwise application and development in horizontal developing chamber (sandwich configuration) gave the best results. Detection was performed using vanillin-H3PO4 reagent. Four new developing solvent systems were proposed: water, 1-propanol-water (20:80, v/v), 1-propanol-water-acetic acid (4:2:1, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v), and at least two of them were needed for the differentiation between all eight compounds. Surprisingly, water enabled the separation of epimers C from EC and GC from EGC, as well as the dimers procianidin B1 and B2. Additionally, C, EGC, B1 and B2 were separated from all the other compounds. The best choice for developing solvent is given for each of the studied compounds. The best separation of the five main catechins (EC, GC, EGC, ECg, EGCg) present in green tea extract was achieved using 1-propanol-water-acetic acid (20:80:1, v/v). The chromatograms of oak bark extract developed in solvents with higher water content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v)) showed less bands than chromatograms developed in solvents with higher organic modifier content (e.g. 1-propanol-water-acetic acid (4:2:1, v/v)). It was proved that such behavior was due to the presence of procyanidins beside the main component catechin.

Cellulose↗

PubMed2005

Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns.

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.

Carboxylic Ester Hydrolases↗

PubMed2004

Antimicrobial and antioxidative enrichment of oak (Quercus robur) bark by rotation planar extraction using ExtraChrom.

The multifunctional ExtraChrom instrument was used in the extraction of antimicrobial and radical scavenging components from oak (Quercus robur L.) bark. Milled and sieved oak bark was extracted with 80% (v/v) methanol solution in water on the ExtraChrom instrument using step-gradient in the preparative separation. Extracts were tested using agar diffusion method on Staphylococcus aureus, Enterobacter aerogenes and Candida albicans. Some extracts showed moderate bactericidal, fungicidal, bacteriostatic and fungistatic activity. The composition related to activity of the fractions and extracts was screened simultaneously by thin-layer chromatography (TLC) detected by UV and by spraying the plate with radical scavenging reagent 1,1-diphenyl-2-picrylhydrazyl (DPPH) to detect antioxidant activity. Thus, we could demonstrate the antiradical and antimicrobial activity of oak beneficial in the storage of wine against the oxidation and human microbial exposure.

Bacteria↗

PubMed2003

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers.

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate-formic acid-water (85:10:15, v/v/v) and n-hexane-ethyl acetate-formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis. Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr = 562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).

Asteraceae↗

PubMed2003

Rotation planar extraction and rotation planar chromatography of oak (Quercus robur L.) bark.

The versatile novel instrument for rotation planar extraction and rotation planar chromatography was exploited for the investigation of oak bark (Quercus robur L.). The same instrument enabled extraction of the bark, analytical proof of (+)-catechin directly in the crude extract and also its fractionation. Additionally, epimeric flavan-3-ols, (+)-catechin and (-)-epicatechin were separated by analytical ultra-micro rotation planar chromatography on cellulose plates with pure water as developing solvent. A comparison of the extraction of oak bark with 80% aqueous methanol by rotation planar extraction and medium pressure solid-liquid extraction was carried out and both techniques were shown to be suitable for the efficient extraction of oak bark. The raw extracts and fractions on thin-layer chromatography showed many compounds that possessed antioxidant activity after spraying with 1,1-diphenyl-2-picrylhydrazyl. Rotation planar fractionation of 840 mg of crude oak bark extract on silica gel gave 6.7 mg of pure (+)-catechin in one run.

Chromatography, Liquid↗

PubMedDate not supplied

Uncertainty in quantitative thin-layer chromatography.

Globalization forces analysts to demand extended control of variability in analytical measurements. A calculation procedure named the "error budget model" following recommendations proposed more than 20 years ago by the Bureau International des Poids et Mesures is established as a rule for evaluating and expressing the measurement uncertainty across a broad spectrum of measurements. This metrological approach common in physical measurement is not applicable in separation techniques and cannot quantitate measurement uncertainty. Our experiments show that it can be used as a planning tool in the validation of thin-layer chromatographic (TLC) methods. A computer program that quantitates uncertainty components associated with potential sources of uncertainty in quantitative TLC is prepared and tested with experimental data. TLC plates with different qualities of stationary phases (TLC and high-performance TLC) spotted with different types of samples are measured. Application is performed manually and automatically. Plates are scanned with UV-vis scanners and a video documentation system in remission and transmission mode and fluorescence. Although the calculated values are close to the values obtained with validation procedures, the error budget approach cannot substitute validation. Calculated results can predict critical points in real quantitative TLC, but they cannot confirm the validity of a selected chromatographic procedure.

Journal Article↗

PubMedDate not supplied

Development and validation of a thin-layer chromatographic method for determination of chloramphenicol residues on pharmaceutical equipment surfaces.

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane-ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n=6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4 degrees C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.

Calibration↗