Determination of Phloridzin and Other Phenolic Compounds in Apple Tree Leaves, Bark, and Buds Using Liquid Chromatography with Multilayered Column Technology and Evaluation of the Total Antioxidant Activity.

Apples are known to be a rich source of phenolic compounds, however detailed studies about their content in the individual parts of apple trees are reported rarely. For this purpose, we tested various stationary phases for the determination of phenolic compounds in leaves, bark, and buds. Phloridzin, phloretin, chlorogenic acid, rutin, and quercitrin were analyzed with high performance liquid chromatography coupled with diode array detection. A YMC Triart C18-ExRS 150 × 4.6 mm, 5 µm particle size analytical column with multilayered particle technology was used. The separation was performed with a mobile phase that consisted of acetonitrile and 0.1% phosphoric acid, according to the gradient program, at a flow rate of 1 mL/min for 12.50 min. The concentration of phenolic compounds from 13 cultivars was in the range of 64.89-106.01 mg/g of dry weight (DW) in leaves, 70.81-113.18 mg/g DW in bark, and 100.68-139.61 mg/g DW in buds. Phloridzin was a major compound. The total antioxidant activity was measured using flow analysis and the correlation with the total amount of phenolic compounds was found. This finding can lead to the re-use of apple tree material to isolate substances that can be utilized in the food, pharmaceutical, or cosmetics industries.

Content of phenolic compounds and antioxidant capacity in fruits of apricot genotypes.

Research on natural compounds is increasingly focused on their effects on human health. In this study, we were interested in the evaluation of nutritional value expressed as content of total phenolic compounds and antioxidant capacity of new apricot (Prunus armeniaca L.) genotypes resistant against Plum pox virus (PPV) cultivated on Department of Fruit Growing of Mendel University in Brno. Fruits of twenty one apricot genotypes were collected at the onset of consumption ripeness. Antioxidant capacities of the genotypes were determined spectrometrically using DPPH• (1,1-diphenyl-2-picryl-hydrazyl free radicals) scavenging test, TEAC (Trolox Equivalent Antioxidant Capacity), and FRAP (Ferric Reducing Antioxidant Power)methods. The highest antioxidant capacities were determined in the genotypes LE-3228 and LE-2527, the lowest ones in the LE-985 and LE-994 genotypes. Moreover, close correlation (r = 0.964) was determined between the TEAC and DPPH assays. Based on the antioxidant capacity and total polyphenols content, a clump analysis dendrogram of the monitored apricot genotypes was constructed. In addition, we optimized high performance liquid chromatography coupled with tandem electrochemical and spectrometric detection and determined phenolic profile consisting of the following fifteen phenolic compounds: gallic acid, 4-aminobenzoic acid, chlorogenic acid, ferulic acid, caffeic acid, procatechin, salicylic acid, p-coumaric acid, the flavonols quercetin and quercitrin, the flavonol glycoside rutin, resveratrol, vanillin, and the isomers epicatechin, (-)- and (+)- catechin.

Silver(I) ions ultrasensitive detection at carbon electrodes-analysis of waters, tobacco cells and fish tissues.

We used carbon paste electrodes and a standard potentiostat to detect silver ions. The detection limit (3 Signal/Noise ratio) was estimated as 0.5 µM. A standard electrochemical instrument microanalysis of silver(I) ions was suggested. As a working electrode a carbon tip (1 mL) or carbon pencil was used. Limits of detection estimated by dilution of a standard were 1 (carbon tip) or 10 nM (carbon pencil). Further we employed flow injection analysis coupled with carbon tip to detect silver(I) ions released in various beverages and mineral waters. During first, second and third week the amount of silver(I) ions releasing into water samples was under the detection limit of the technique used for their quantification. At the end of a thirteen weeks long experiment the content of silver(I) ions was several times higher compared to the beginning of release detected in the third week and was on the order of tens of nanomoles. In subsequent experiments the influence of silver(I) ions (0, 5 and 10 µM) on a plant model system (tobacco BY-2 cells) during a four-day exposition was investigated. Silver(I) ions were highly toxic to the cells, which was revealed by a double staining viability assay. Moreover we investigated the effect of silver(I) ions (0, 0.3, 0.6, 1.2 and 2.5 µM) on guppies (Poecilia reticulata). Content of Ag(I) increased with increasing time of the treatment and applied concentrations in fish tissues. It can be concluded that a carbon tip or carbon pencil coupled with a miniaturized potentiostat can be used for detection of silver(I) ions in environmental samples and thus represents a small, portable, low cost and easy-to-use instrument for such purposes.

Study of Interactions between Metallothionein and Cisplatin by using Differential Pulse Voltammetry Brdickás reaction and Quartz Crystal Microbalance.

Treatment strategies for tumour diseases are progressively focusing on personalization of medicine. However, this focus requires methods revealing the early general biological mechanisms, including the formation anti-cancer drugs' resistance. The low molecular mass protein metallothionein is thought to be the crucial for the formation of resistance in tumour treatment based on the platinum-cytostatics. The interactions between metallothionein (MT) and cisplatin were determined by the adsorptive transfer stripping technique coupled with the differential pulse votlammetry Brdickás reaction. The signals related to the MT-cisplatin complex appeared at -0.9 V. The formation of this complex depended on the time of interaction between cisplatin and MT. The complex formation was consequently confirmed by quartz crystal microbalance analyses. The formation of this complex was detectable even after a 20 s long interaction. Moreover, we detected presence of MT-cisplatin complex in the blood of male rats treated with this drug.

Determination of Vitamin C (Ascorbic Acid) Using High Performance Liquid Chromatography Coupled with Electrochemical Detection.

Vitamin C (ascorbic acid, ascorbate, AA) is a water soluble organic compound that participates in many biological processes. The main aim of this paper was to utilize two electrochemical detectors (amperometric - Coulouchem III and coulometric - CoulArray) coupled with flow injection analysis for the detection of ascorbic acid. Primarily, we optimized the experimental conditions. The optimized conditions were as follows: detector potential 100 mV, temperature 25 °C, mobile phase 0.09% TFA:ACN, 3:97 (v/v) and flow rate 0.13 mL·min-1. The tangents of the calibration curves were 0.3788 for the coulometric method and 0.0136 for the amperometric one. The tangent of the calibration curve measured by the coulometric detector was almost 30 times higher than the tangent measured by the amperometric detector. Consequently, we coupled a CoulArray electrochemical detector with high performance liquid chromatography and estimated the detection limit for AA as 90 nM (450 fmol per 5 µL injection). The method was used for the determination of vitamin C in a pharmaceutical preparations (98 ± 2 mg per tablet), in oranges (Citrus aurantium) (varied from 30 to 56 mg/100 g fresh weight), in apples (Malus sp.) (varied from 11 to 19 mg/100 g fresh weight), and in human blood serum (varied from 38 to 78 µM). The recoveries were also determined.

Electrochemical Determination of the Antioxidant Potential of Some Less Common Fruit Species.

Various berries and fruit types of less common fruit species are known to contain antioxidants. Consumption of high amounts of antioxidant flavonoids, which display a variety of biological properties, including antiproliferative and anti-inflammatory activity, may have a positive impact on human health, particularly for the prevention of cancer and other inflammatory diseases. In these studies, based on the hypothesis that the fruit extract with the highest content would possess significantly higher health benefits, flavonoid-rich extracts were obtained from some less common fruit species - Blue Honeysuckles (Lonicera Kamtschatica and Lonicera edulis, Turcz. ex. Freyn), Saskatoon berry (Amelanchier alnifolia Nutt.) and Chinese Hawthorn (Crataegus pinnatifida BUNGE) - grown from germplasm held at the Mendel University of Agriculture and Forestry in Brno, Czech Republic and then characterized in terms of biological value based on the results from a relative antioxidant capacity assessment. The antioxidant content evaluation was based on the total flavonoid amount, determined by liquid chromatography with electrochemical detection (HPLC-ED). A DPPH• test was applied as a reference. The antioxidant content measured in Chinese Hawthorn fruit extract identified it as a potent source of flavonoid antioxidants, with a content 9-fold higher than that seen in Amelanchier fruit. The multifunctional HPLC-ED array method coupled with a DPPH• reference appears to be the optimal analytical progress, accurately reflecting the nutritivetherapeutic properties of a fruit.

Lactoferrin Isolation Using Monolithic Column Coupled with Spectrometric or Micro-Amperometric Detector.

Lactoferrin is a multifunctional protein with antimicrobial activity and others tohealth beneficial properties. The main aim of this work was to propose easy to usetechnique for lactoferrin isolation from cow colostrum samples. Primarily we utilizedsodium dodecyl sulphate - polyacrylamide gel electrophoresis for isolation of lactoferrinfrom the real samples. Moreover we tested automated microfluidic Experionelectrophoresis system to isolate lactoferrin from the collostrum sample. The welldeveloped signal of lactoferrin was determined with detection limit (3 S/N) of 20 ng/ml. Inspite of the fact that Experion is faster than SDS-PAGE both separation techniques cannotbe used in routine analysis. Therefore we have tested third separation technique, ionexchange chromatography, using monolithic column coupled with UV-VIS detector (LCUV-VIS). We optimized wave length (280 nm), ionic strength of the elution solution (1.5M NaCl) and flow rate of the retention and elution solutions (0.25 ml/min and 0.75 ml/min.respectively). Under the optimal conditions the detection limit was estimated as 0.1 µg/mlof lactoferrin measured. Using LC-UV-VIS we determined that lactoferrin concentrationvaried from 0.5 g/l to 1.1 g/l in cow colostrums collected in the certain time interval up to 72 hours after birth. Further we focused on miniaturization of detection device. We testedamperometric detection at carbon electrode. The results encouraged us to attempt tominiaturise whole detection system and to test it on analysis of real samples of humanfaeces, because lactoferrin level in faeces is closely associated with the inflammations ofintestine mucous membrane. For the purpose of miniaturization we employed thetechnology of printed electrodes. The detection limit of lactoferrin was estimated as 10µg/ml measured by the screen-printed electrodes fabricated by us. The fabricatedelectrodes were compared with commercially available ones. It follows from the obtainedresults that the responses measured by commercial electrodes are app. ten times highercompared with those measured by the electrodes fabricated by us. This phenomenonrelates with smaller working electrode surface area of the electrodes fabricated by us(about 50 %) compared to the commercial ones. The screen-printed electrodes fabricatedby us were utilized for determination of lactoferrin faeces. Regarding to fact that sample offaeces was obtained from young and healthy man the amount of lactoferrin in sample wasunder the limit of detection of this method.

Use of liquid chromatography with electrochemical detection for the determination of antioxidants in less common fruits.

Neurodegenerative disorders (NDD) have become the common global health burden over the last several decades. According to World Health Organization (WHO), a staggering 30 million people will be affected by Alzheimer's disease in Europe and the USA by 2050. Effective therapies in this complex field considering the multitude of symptoms associated with NDD indications, have not been found yet. Based on the results of NDD related studies, prevention appears to be the promise alternative. Antioxidative and anti-inflammatory properties are hypothesized for natural phenolics, a group of plant secondary products that may positively impact neurodegenerative diseases. In these studies, phenolic-rich extracts from less common fruit species: Blue honeysuckle (Lonicera edulis, Turcz. ex. Freyn), Saskatoon berry (Amelanchier alnifolia Nutt.), and Chinese hawthorn (Crateagus pinnatifida Bunge) were obtained and analyzed to detect neuroprotective substances content and establish a potential therapeutic value. High performance liquid chromatography with electrochemical detection was optimized and further applied on analysis of the extracts of less common fruit species. It was observed that Chinese hawthorn and Blue honeysuckle extracts are potent source of neuroprotective phenolic antioxidants. In accordance the results, it appears that the fruit or formulated products may have the potential for the prevention of neurodegenerative diseases.

Electrochemical determination of Ag-ions in environment waters and their action on plant embryos.

We utilized liquid chromatography coupled with electrochemical detector (HPLC-ED) for analyzing of silver ions. The optimization of basic chromatographic parameters has been done. The detection limit (3 S/N) obtained were 20 nmol/dm(3). Influence of different interferences (anions and cations) on current response of silver ions has been described. Moreover, we used HPLC-ED to analyze waters of different purity including photographic emulsion, which naturally contained silver ions. We found out that content of silver ions in the emulsion was 1.57 x 0.03 mmol/dm(3). Moreover, we investigated influence of silver ions on early somatic embryos of Blue Spruce. We were interested in the issue how much silver ions can embryos uptake during four days long treatment. For this purpose, we used optimized HPLC-ED technique. The content increased with increasing treatment time and applied concentration. We also studied how silver ions can influence thiols content in the treated embryos. For these purposes we used adsorptive transfer stripping voltammetry in connection with differential pulse voltammetry--Brdicka reaction. It clearly follows from the obtained results that content of thiols increased with increasing treatment time and applied concentration.

Utilizing of Square Wave Voltammetry to Detect Flavonoids in the Presence of Human Urine.

About biological affecting of flavonoids on animal organisms is known less,thus we selected flavonoids, flavanones and flavones, and their glycosides, which wereexamined as potential inducers of cytochrome(s) P450 when administrated by gavages intoexperimental male rats. The study was focused on induction of CYP1A1, the majorcytochrome P450 involved in carcinogen activation. The data obtained demonstrate thenecessity of taking into account not only ability of flavonoids to bind to Ah receptor(induction factor) but also to concentrate on their distribution and metabolism (includingcolon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than µM. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).

Toxicological aspects of flavonoid interaction with biomacromolecules.

OBJECTIVES: Flavonoids are widely accepted as health promoting phytochemicals, however, some flavonoids show ability of direct interaction with DNA and/or enhance carcinogen activation into DNA modifying agents. Thus, their potential harmful effect on the human body should be examined in detail. METHODS: Direct interaction of flavonoids (quercetin, rutin) with DNA was examined using square wave voltammetry on carbon paste electrode. Induction effect of selected flavonoids on content of cytochrome P450 1A1, carcinogens activating enzyme, in colon and liver microsomal samples of animals exposed to flavonoids was determined by Western blotting, using anti-cytochrome P450 1A1 specific antibody. RESULTS: Of the natural flavonoids tested, induction of CYP1A1 was elicited by the typical citrus flavonoid naringenin in the colon, as well as by flavone in the liver. Moreover, synthetic beta-naphthoflavone and naturally occurring chrysin, quercetin and diosmin induced CYP1A1 in both tissues. The oxidation signals of guanine and adenine in the DNA molecule were decreased in the presence of flavonoids. CONCLUSIONS: Although flavonoids are often considered to be safe because of their "plant origin", ingestion of flavonoids should be taken with caution. Enhanced expression of CYP1A1 in colon tissue might be responsible for increasing incidence of colorectal carcinoma in humans. Electrochemistry can be used to study the interactions of flavonoids and DNA.

High-performance liquid chromatographic method with amperometric detection employing boron-doped diamond electrode for the determination of sildenafil, vardenafil and their main metabolites in plasma.

A simple, fast and sensitive HPLC method with electrochemical detection employing boron-doped diamond electrode (BDD) for the determination of sildenafil (Viagra™), vardenafil (Levitra™) and their main metabolites, N-desmethyl sildenafil and N-desethyl vardenafil in human plasma is presented. The assay involved drug extraction by tert-butyl methyl ether and isocratic reversed-phase liquid chromatography with amperometric detection. Complete separation of all analytes was achieved within 12 min. The mobile phase consisted of 20mM sodium dihydrogen phosphate with 40 mM sodium perchlorate/acetonitrile (70:30, v/v), pH 3.5. The electrode working potential was +1520 mV (vs. Pd/H(2)). Calibration curves were linear over the concentration range of 10-400 ng mL(-1). Phloretin was used as an internal standard. The limits of detection (LOD) and quantification (LOQ) for the studied analytes were within the range of 2-4 ng mL(-1) and 7.0-13.4 ng mL(-1), respectively. The developed method was applied to human plasma samples spiked with analytes at therapeutic concentrations. The study confirms the method's suitability for both pharmacokinetic studies and therapeutic monitoring.

Comparison of UV and charged aerosol detection approach in pharmaceutical analysis of statins.

CAD (charged aerosol detector) has recently become a new alternative detection system in HPLC. This detection approach was applied in a new HPLC method for the determination of three of the major statins used in clinical treatment-simvastatin, lovastatin and atorvastatin. The method was optimized and the influence of individual parameters on CAD response and sensitivity was carefully studied. Chromatography was performed on a Zorbax Eclipse XDB C18 (4.6 mm x 75 mm, 3.5 microm), using acetonitrile and formic acid 0.1% as mobile phase. The detection was performed using both CAD (20 pA range) and DAD (diode array detector-238 nm) simultaneously connected in series. In terms of linearity, precision and accuracy, the method was validated using tablets containing atorvastatin and simvastatin. The CAD is designated to be a non-linear detector in a wide dynamic range, however, in this application and in the tested concentration range its response was found to be perfectly linear. The limits of quantitation (0.1 microg/ml) were found to be two times lower than those of UV detection.

Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection.

Easy, efficient and low demanding separation of mRNA from biological material is needed to study gene expression and to use in chip technologies. It is common knowledge that each mRNA molecule contains sequence of 25 adenines. This feature can be used for binding mRNA on the surface of the particles coated by thymine chains. The present work reports on suggesting and optimizing of mRNA separation and detection from biological material via paramagnetic microparticles coupled with electrochemical detection. Primarily we optimized cyclic and square wave voltammetric conditions to detect poly(A), which was used as standard to mimic behaviour of mRNA. Under the optimized square wave voltammetric conditions (frequency 280 Hz, accumulation time 200 s, supporting electrolyte and its temperature: acetate buffer 4.6 and 35 degrees C) we estimated detection limit down to 1 ng of poly(A) per ml. To enhance effectiveness and repeatability of isolation of nucleic acid automated approach for rinsing and hybridizing was proposed. We optimized the whole procedure and experimental conditions. Using automated way of isolation and under optimized conditions the yield of poly(A) (isolated concentration of poly(A)/given concentration of poly(A)*100) was approximately 75%. The suggested and optimized method for poly(A) isolation and detection was utilized for the analysis of brain tissues of patients with traumatic brain injury. The total amount of isolated mRNA varied from 40 to 760 g of mRNA per g of brain tissue. The isolation of mRNA from six samples per run was not longer than 2.5h. Moreover, we applied the optimized procedure on fully automated pipetting instrument to isolate mRNA. The instrument was successfully tested on the analysis of extracts from roots of maize plants treated with cadmium(II) ions.

RP-HPLC analysis of phenolic compounds and flavonoids in beverages and plant extracts using a CoulArray detector.

Methods were developed for the analysis of natural antioxidants including phenolic compounds and flavonoids in beverages and plant extracts using gradient HPLC with multi-channel electrochemical coulometric detection. Suitability of various reversed-phase columns for this purpose was compared; pH and mobile phase gradients were optimized with respect to the separation selectivity and sensitivity of detection. Because of different target compounds in various sample types, the overlapping resolution maps and the normalized resolution product approaches described earlier were used to select optimum columns and gradients to suit the analysis of the individual sample types. The methods were applied to the analysis of phenolic compounds and flavonoids in beer, wine, tea, and yacon extracts. 32 phenolic compounds were identified and determined, including derivatives of benzoic and cinnamic acids, flavones, and a few related glycosides. Eight-channel CoulArray detection offers high selectivity and sensitivity with limits of detection in the low microg L(-1) range, at least an order of magnitude lower than single-channel coulometric detection using the Coulochem detector. No special sample pretreatment is necessary and, because of the compatibility of the CoulArray detector with gradient elution, phenolic antioxidants of different polarities can be determined in a single run. In addition to the retention times, the ratios of the areas of the pre-dominant and post-dominant peaks to the area of the dominant peak can be used for improved identification of natural antioxidants.