Purity assessment of recombinant human granulocyte colony-stimulating factor in finished drug product by capillary zone electrophoresis.

Current methods for determination of impurities with different charge-to-volume ratio are limited especially in terms of sensitivity and precision. The main goal of this research was to establish a quantitative method for determination of impurities with charges differing from that of recombinant human granulocyte colony-stimulating factor (rhG-CSF, filgrastim) with superior precision and sensitivity compared to existing methods. A CZE method has been developed, optimized, and validated for a purity assessment of filgrastim in liquid pharmaceutical formulations. Optimal separation of filgrastim from the related impurities with different charges was achieved on a 50 µm id fused-silica capillary of a total length of 80.5 cm. A BGE that contains 100 mM phosphoric acid adjusted to pH 7.0 with triethanolamine was used. The applied voltage was 20 kV while the temperature was maintained at 25°C. UV detection was set to 200 nm. Method was validated in terms of selectivity/specificity, linearity, precision, LOD, LOQ, stability, and robustness. Linearity was observed in the concentration range of 6-600 µg/mL and the LOQ was determined to be 0.3% relative to the concentration of filgrastim of 0.6 mg/mL. Other validation parameters were also found to be acceptable; thus the method was successfully applied for a quantitative purity assessment of filgrastim in a finished drug product.

Interference of selected flavonoid aglycons in platelet aggregation assays.

BACKGROUND: Flavonoids are widely distributed across the plant kingdom and are therefore common ingredients in an everyday diet. Some flavonoids have a potential to affect platelet aggregation; most often antiaggregatory effects of flavonoids are observed. The objective of this research was to evaluate the in vitro effect of a selected set of flavonoids on platelet aggregation in whole blood. METHODS: The effect of five selected flavonoids (pinocembrin-7-methylether, epicatechin, hesperetin, 6-hydroxyflavone and 3,6-dihydroxyflavone) on platelet aggregation was studied in the citrated whole blood samples collected from 75 healthy volunteers. A Multiplate(®) impedance analyzer and five different aggregation inducers (ADP, arachidonic acid, collagen, ristocetin and TRAP-6) were utilized for the analysis of samples. RESULTS: Minimal antiaggregatory concentrations (MINaAC) of flavonoids in individual tests were reported in the following ranges: 0.12-1.91 µM; 15.26-244.14 µM; 15.26-122.07 µM; and 0.06-15.26 µM for ADP, collagen, TRAP-6 and ristocetin aggregation-inducers, respectively. When arachidonic acid was used for induction of platelet aggregation, a proaggregatory effect was observed for pinocembrin-7-methylether, epicatechin, hesperetin and 3,6-dihydroxyflavone, while the expected antiaggregatory effect was observed only for 6-hydroxyflavone (MINaAC=7.63 µM). CONCLUSIONS: Flavonoids interfere with in vitro platelet aggregation assays exhibiting either anti- or proaggregatory effects in concentration ranges that can be achieved in circulation by dietary intake. Thus, dietary intake of flavonoids should be taken into account when interpreting the results of whole blood platelet aggregation.

Evaluation of antiaggregatory activity of flavonoid aglycone series.

BACKGROUND: Among natural compounds, present in every day diet, flavonoids have shown beneficial effect in prevention of cardiovascular diseases that can be attributed, at least partially to the described antiaggregatory activity i.e. antiplatelet effects of flavonoids. Due to the ever increasing pharmacological interest in antiplatelet agents a systematic experimental evaluation of large flavonoid series is needed. METHODS: A set of thirty flavonoid aglycones has been selected for the evaluation. All measurements of aggregation were done under standardized and firmly controlled in vitro conditions. The whole blood samples, multiple platelet functional analyzer and adenosine diphosphate (ADP) as a weak agonist of aggregation were selected for this purpose. RESULTS: The results were expressed as minimal concentration of flavonoid that can significantly lower the platelet aggregation compared to the corresponding untreated sample (minimal antiaggregatory concentration--MINaAC). All analyzed flavonoids exhibited antiaggregatory activity MINaAC ranging from 0.119 µM to 122 µM, while the most potent representatives were 3,6-dihydroxyflavone (0.119 µM) and syringetin (0.119 µM). CONCLUSIONS: Measurable antiplatelet activity established at submicromolar flavonoid concentrations suggests that even a dietary consumption of some flavonoids can make an impact on in vivo aggregation of platelets. These findings also point out a therapeutical potential of some flavonoids.

Recent advances in the application of high performance liquid chromatography in the analysis of polyphenols in wine and propolis.

Wine and propolis are among the richest sources of polyphenolics. Flavonoids and phenolic acids are main pharmacological components whose benefits have been shown in numerous reports. This paper presents a review of the latest applications of HPLC for determination of polyphenolics in propolis and wine. Special attention is focused on sample preparation, chromatographic conditions, and detection of polyphenolics. HPLC will undoubtedly be the most used method that is readily available for analysis, as well as for isolation of polyphenolics in complex mixtures.

Effect of natural biostimulants on yield and nutritional quality: an example of sweet yellow pepper (Capsicum annuum L.) plants.

BACKGROUND: Modifications in growing techniques can affect the yield and nutritional quality of various cultivated plant species. Owing to its high nutritional value, pepper (Capsicum annuum L.) was used in this study as a model plant to investigate the effect of natural biostimulants on yield and fruit quality parameters under conditions of reduced fertilisation. RESULTS: A positive influence of biostimulant treatment on yield parameters was observed. The overall increase in the pigment content of leaves after biostimulant application agreed well with the higher total and commercial yields of treated pepper cultivars compared with their controls. The results showed that natural biostimulants had a positive effect on the vitamin C and total phenolic contents in pepper fruits during the hot summer season. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) antioxidant activities were also significantly higher (P < 0.05) in treated plants and correlated strongly with all measured quality parameters except total phenolic content. CONCLUSION: Generally, biostimulants improved the antioxidant activity, vitamin C and phenolic contents in fruits as well as the pigment content in leaves of treated compared with non-treated pepper plants grown hydroponically. Thus the application of biostimulants could be considered as a good production strategy for obtaining high yields of nutritionally valuable vegetables with lower impact on the environment.

From functional food to medicinal product: systematic approach in analysis of polyphenolics from propolis and wine.

In the last decade we have been working on standardization of propolis extract and determination of active constituents of wine those are rich in polyphenolics and have nutritional as well as therapeutic value. Here we are summarizing our results and providing overview on systematic approach how to analyse natural products rich in flavonoids and phenolic acids.Chromatographic methods (thin layer chromatography and high performance liquid chromatography) were used for identification, quantification, and characterization of individual flavonoid or phenolic acid. Total content of active constituents and antioxidant activity were determined by spectrophotometry. Pharmacokinetic parameters were determined by high performance liquid chromatography and using appropriate software. Quantitative structure-activity relationship study of antioxidant activity was conducted, as well as assessment of prolonged propolis supplementation on antioxidative status of organism.Thin layer chromatography-densitometry has been proven as quick and reliable method for standard analysis of propolis and wine; the best mobile phase being chloroform - methanol - formic acid (98-100%) in ratio 44 : 3.5 : 2.5 (v/v). Higher number of polyphenolics was determined by high performance liquid chromatography; 15 compared to 9 by thin layer chromatography. Interactions in situ with acetylsalicylic acid were detected with most of polyphenolics analysed. Plasma protein binding and blood-barrier penetration was greatest for flavone. The interactions with human serum albumin have been grater than 95% for all flavonoids analysed. The prolonged propolis consumption increased superoxide dismutase activity.The necessity of standardization of natural products and their registration as functional nutraceuticals demand easy, quick and inexpensive methods of analysis. In this work we provided overview of analytical part for polyphenolics that could be used as data for possible registration of final products either as functional food or medicinal product.This feature introduces the readers to the authors' research through a concise overview of the selected topic. Reference to important work from others in the field is included.

The synthesis and antimicrobial activity of some 4-hydroxycoumarin derivatives.

Due to exceptional reactivity of 4-hydroxycoumarin, the synthesis of new coumarin derivatives of dimer and tetramer type has been carried out. The synthesis was carried out from 4-hydroxycoumarin and various aromatic aldehydes. In this way, compounds of the dimer 3,3'-(benzilidene)bis (4-hydroxycoumarin) type, as well as of the tetramer 3,3',3'',3'''-(1,4-dimethylenphenyl)tetra (4-hydroxycoumarin) type were prepared. The newly synthesized derivatives contain different functional groups, and as such they could exhibit microbiological activity. Therefore, we tested the microbiological activity of these derivatives on various species of bacteria and fungi. The tested compounds have shown different activity in terms of growth inhibition of microorganisms. Newly synthesized derivatives exhibit antibacterial activities, manifested as growth inhibition on Gram-positive bacteria types (Bacillus, Staphylococcus), while the activity against Candida was much weaker. The same compound did not show any antimicrobial activity against two Gram-negative bacteria types (Escherichia coli, Pseudomonas aeruginosa). The compound 1 showed the best microbiological activity. The obtained results confirmed its good antibacterial and antimycotic activities against different microorganisms.

In vivo study of propolis supplementation effects on antioxidative status and red blood cells.

In vivo study has been conducted on 47 healthy women and men in order to investigate whether daily intake of powdered propolis extract during 30 days has any influence on the following blood parameters: activity of superoxide dismutase, glutathione peroxidase and catalase, concentration of plasma malondialdehyde, total cholesterol, low- and high-density lipoprotein cholesterol, triglycerides, glucose, uric acid, ferritin and transferrin, together with routine red blood cell parameters. The effect of daily propolis intake seems to be time and gender related. For the men test group after the initial 15 days of propolis treatment, 23.2% (p=0.005) decrease in concentration of malondialdehyde was observed. After 30 days of treatment, statistically significant (p=0.010) 20.9% increase in superoxide dismutase activity and change in some of the red blood cell parameters were detected. For the women test group, the propolis treatment did not induce a change in any of the measured parameters.

Evaluation of antioxidative activity of Croatian propolis samples using DPPH* and ABTS*+ stable free radical assays.

Propolis is one of the richest sources of plant phenolics (flavonoids and phenolic acids), which are widely recognized as rather strong antioxidants. The aim of our work was to use colored stable free radical (DPPH* and ABTS*+) spectrophotometric and thin-layer chromatographic (TLC) assays to study the antioxidative behavior of the phenolics (caffeic acid, galangin and pinocembrin) most commonly present in Croatian propolis samples obtained from different Croatian regions. We propose a mathematical model providing a more sophisticated interpretation of the obtained results and a new parameter named antioxidative efficiency (AOE) is introduced. The kinetic behaviour of chosen standards determined by spectrophotometric assays follows the exponential decrease of the absorption curve. Explained numerically, AOE represents the absolute value of the first derivative of an absorbance curve in the point A0/e (where A0 is the absorbance measured at t = 0 and e is the natural logarithm base). The advantage of this newly introduced parameter is that it provides an easy and accurate mutual comparison between the rates of antioxidative efficiency of different propolis samples. A TLC assay was only applicable in the case of the DPPH* radical. Dose-response curves were described using a linear function with AOE expressed as a coefficient of the slope. The chromatographic method was shown to be very rapid, reliable and easy-to-perform.

Evaluation of novel sample identification approach based on chromatographic fingerprint set correlation homogeneity analysis.

Instead of usual rationale for chromatographic fingerprint based sample identification which relies upon visual inspection or principal component analysis of raw or aligned chromatograms novel nonparametric statistical measure of fingerprint set homogeneity is proposed. Randomization test is applied for significance analysis of fingerprint set homogeneity while average maximum crosscorrelation is used as a merit function. Chromatogram sets generated by random selection from standard and unknown sample chromatogram collections are compared with respect to merit function values with set of chromatograms that represents standard and/or unknown sample. In that instance fingerprint homogeneity significance is represented by the fraction of random chromatogram sets that have higher merit values than the standard and/or unknown sample sets. A set of peptide maps corresponding to different haemoglobin variants has been selected for evaluation of proposed test. This approach is compared to chromatogram alignment based on correlation optimized warping coupled with principal component or cluster analysis. Proposed method is simple i.e. straightforward sample identification procedure which reliability has been evaluated here. Impact of this approach on peptide mapping validation and system suitability analysis is discussed.

Lipophilicity study of salicylamide.

Molecular lipophilicity was studied using salicylamide as a model drug. Log P value for the target compound was experimentally determined by the shake-flask method and calculated using nine different computer programs based on atom/fragment contributions, structural parameters, atom-type electrotopological-state indices and neural network modeling, or topological structure descriptors. Our analysis demonstrates good agreement between the experimentally observed log P value of salicylamide and the value calculated by the CSLogP program, based on topological structure descriptors and electrotopological indices.

A comparison of the nutritional value and food safety of organically and conventionally produced wheat flours.

Growing interest in organic agriculture has prompted this study aiming to evaluate nutritional content of wheat flours originating from organic and conventional production systems. Obtained results showed that organic samples had significantly lower protein content and lower levels of Ca, Mn and Fe compared to conventional samples. Protein digestibility and levels of K, Zn and Mo were significantly higher in organic than in conventional wheat flours. Regarding undesirable metals, significantly higher levels of As and Cd were found in conventional compared to organic wheat flours. Although the mean concentrations of zearalenone and ochratoxin A were higher in conventional than in organic flours, this difference was not significant. This study revealed that organic agriculture has the potential to yield products with some relevant improvements in terms of high quality proteins and microelements contents, while the reduction in contamination with toxic elements and mycotoxins may be accomplished.

Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis is a resinous hive product collected by honeybees from various plant sources. It is widely used in traditional medicine and is reported to have a broad spectrum of pharmacological effects (antibacterial, antihepatoxic, antioxidative, anti-inflammatory, etc.). Thus the aim of this study was to assess cytotoxic effect of various ethanol propolis extractions on the cervical tumor cell line (HeLa) and compare it with their phenolic acids and flavonoids composition. MATERIALS AND METHODS: Twenty samples of raw propolis were collected from 17 localities of Croatia (I-XVII), 2 of Bosnia and Hercegovina (XVIII, XIX) and 1 of Macedonia (XX). Reverse phase HPLC was used to determine the chemical composition of polyphenols. Biological experiments were carried out in vitro on cervix adenocarcinoma cell line (HeLa). RESULTS: Phenolic acids (ferulic acid, p-coumaric acid, caffeic acid) and flavonoids (tectochrysin, galangin, pinocembrin, pinocembrin-7-methylether, chrysin, apigenin, kaempferol, quercetin) have been determined using HPLC analysis in 20 ethanolic propolis extracts. All samples contain tectochrysin in ranges of 0.1988 mg/g (XVIII) to 1.2004 mg/g (III), while caffeic acid and quercetin have not been found. Flavonoid that is most abundant is galangin in ranges from 0.3706 mg/g (XVII) to 47.4879 mg/g (IX). The samples of propolis numbers I, VI and X applied in the investigated concentration range manifested significant reduction of cell growth. GI(50) value as indicator of cytotoxicity among propolis samples showed that propolis number VII is the most effective (GI(50) =76 µg/ml) followed by propolis nos. XV, XVIII and I. CONCLUSION: Antiproliferative and cytotoxic effect of propolis on the HeLa cells is not correlating with the concentration of particular components but on establishing the possible synergistic antiproliferative activity of individual phenolic acid and flavonoids. Differences in the chemical composition lead to diversity in biological activity of propolis samples.

QSAR study of antioxidant activity of wine polyphenols.

Quantitative structure activity relationships (QSAR) were obtained describing the antioxidant activity of the main pharmacologically active polyphenols of wine, using molecular properties and descriptors derived from the 2D and 3D representations of molecules. The best models for the prediction of the ability to scavenge the ABTS radical cation were obtained by polynomial regression analysis using zero-order connectivity index and molar refractivity. Statistically, significant models for lipid peroxidation inhibiting effects of flavonoids were obtained by polynomial and multiple regression using lipophilicity, Balaban index, Balaban-type index and 3D GETAWAY descriptor. The 3D descriptors possess the ability for discrimination of stereoisomers, like catechin and epicatechin. We demonstrated that a size and shape of molecules, as well as steric properties, play an important role in the antioxidant activity of polyphenols.

QSAR study of antimicrobial activity of some 3-nitrocoumarins and related compounds.

A new class of antimicrobial agents, 3-nitrocoumarins and related compounds, has been chosen as a subject of the present study. In order to explore their activity and molecular properties that determine their antimicrobial effects, QSAR models have been proposed. Most of the 64 descriptors used for the development were extracted from semiempirical and density functional theory (DFT) founded calculations. For this study literature data containing results of microbiological activity screening of 33 coumarin derivatives against selected clinical isolates of C. albicans (CA) and S. aureus (SA) have been selected. Multivariate predictive models based on random forests (RF) and two hybrid classification approaches, genetic algorithms (GA) associated with either support vector machines (SVM) or k nearest neighbor (kNN), have been used for establishment of QSARs. An applied feature selection approach enabled two-dimensional linear separation of active and inactive compounds, which was a necessary tool for rational candidate design and descriptor relevance interpretation. Candidate molecules were checked by cross-validated models, and selected derivatives have been synthesized. Their antimicrobial activities were compared to antimicrobial activities of the representative derivatives from the original set in terms of minimal inhibitory concentration (MIC) against chosen SA and CA ATCC strains. High ranking of descriptors consistent with the degree of hydrolytic instability of selected compounds is common to models of antimicrobial activity against both microorganisms. However, descriptor ranking indicates different antimicrobial mechanisms of action of chosen coumarin derivatives against selected microbial species.

Novel approach to evolutionary neural network based descriptor selection and QSAR model development.

Capability of evolutionary neural network (ENN) based QSAR approach to direct the descriptor selection process towards stable descriptor subset (DS) composition characterized by acceptable generalization, as well as the influence of description stability on QSAR model interpretation have been examined. In order to analyze the DS stability and QSAR model generalization properties multiple random dataset partitions into training and test set were made. Acceptability criteria proposed by Golbraikh et al. [J. Comput.-Aided Mol. Des., 17 (2003) 241] have been chosen for selection of highly predictive QSAR models from a set of all models produced by ENN for each dataset splitting. All QSAR models that pass Golbraikh's filter generated by ENN for each dataset partition were collected. Two final DS forming principles were compared. Standard principle is based on selection of descriptors characterized by highest frequencies among all descriptors that appear in the pool [J. Chem. Inf. Comput. Sci., 43 (2003) 949]. Search across the model pool for DS that are stable against multiple dataset subsampling i.e. universal DS solutions is the basis of novel approach. Based on described principles benzodiazepine QSAR has been proposed and evaluated against results reported by others in terms of final DS composition and model predictive performance.