STUDIA CHEMIA - Issue no. 2 T1 / 2019 - Table of contents

Professor Florin Dan Irimie on His 65th Anniversary

Keywords:

Similar to the petroleum industry, biodiesel industry generates unwanted by-products. Biodiesel production generate huge amount of crude glycerol- one part of glycerol is produced at every ten parts of biodiesel, and has a negative influence on the biodiesel price. A solution to reduce the negative environmental problems and the cost of biodiesel is to use crude glycerol as carbon source for microbial growth media in order to produce valuable organic chemicals. Recently, the research group from University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca developed sustainable opportunities for L (+) lactic acid production via R. oryzae NRRL 395 fermentation on biodiesel crude glycerol. The current research aims at improving the production of L-lactic acid using crude glycerol as substrate, immobilized spores and inhibited alcohol dehydrogenase of R. oryzae in discontinuous and semi continuous fermentation processes in order to make the process more efficient. According to several experimental dataset under different process parameters, multiple regression methods were employed to establish mathematical models for prediction of the L lactic acid production. Determination coefficient values states that predicted values were in good agreement with the experimental values. Introducing the fractional order in models lead to a good phenomenological description. The resulted models can be used both in optimization process and prediction instead of time and resource consuming experiments.

Keywords: biodiesel; crude glycerol; process optimization, mathematical model, fractional order model

A Plackett-Burman factorial design of experiments was created to optimize the protocols of preparation of E. coli DH5α competent cells and transformation of these cells by heat shock method using a chiA_pUC57 plasmid. The numerical parameters to be optimized were: the pH, the concentration of CaCl2, the cell concentration of the culture used for the preparation of the competent cells, the temperature of defrosting of the competent cells, the concentration of plasmid DNA. It was also considered a qualitative factor that might influence the transformation efficiency, namely the use of ultrasound in the heat shock step of transformation protocol. A design of experiments based on 26 experimental values was created. Analyzing this experimental setup by both, Plackett-Burman factorial design and surface response design, it was highlighted that the pH, the concentration of calcium chloride and the concentration of plasmid DNA have a significant influence on the transformation efficiency. The optimal conditions for the preparation and transformation of E. coli DH5α competent cells with chiA_pUC57 plasmid where when the pH of a 40 mM CaCl2 solution was 6, the competent stock cells were thawed slowly on ice and in the heat shock step the cells were subjected to ultrasounds treatment.

Keywords: design of experiments; Minitab; Plackett-Burman factorial design; surface response design; competent cell; heat shock transformation

Free radicals and reactive oxygen species are produced in the human body as a part of metabolic processes. Reactive species in low levels are important for cellular activities. Excessive amounts of reactive species can be harmful because they can produce lipid peroxidation, proteins and ADN oxidation. For reduce these harmful effects the organism requires an antioxidant defence. Oxidative stress is involve in atherosclerosis, coronary heart disease, metabolic syndrome, type 2 diabetes mellitus. The aerobic and anaerobic exercises have different effects on the muscles, but both influence positively the biomarkers of oxidative stress. Studies prove that aerobics increase endogenous antioxidant status. Regular moderate exercises produce an increase in antioxidant activity, due to the changes in redox homeostasis. The aim of this review is to discuss the importance of a constant physical activity for increase the body's antioxidant system and to protect against oxidative stress.

Keywords: Free radicals, reactive oxygen species, oxidative stress, metabolic syndrome, aerobic exercises, anaerobic exercises, antioxidant system

Ascorbate oxidase is a well-known copper-containing enzyme however, its biological role and mechanism are still unclear. Reported here is a new protocol for purification of ascorbate oxidase from Cucurbita pepo medullosa (field pumpkin), with improved yields. The enzyme is biochemically characterized, including Michaelis-Menten parameters. Also, purification and characterization of the peroxidase from the same source is described, featuring an unusually high affinity for hydrogen peroxide(low-micromolar).

Keywords: ascorbate oxidase, peroxidase, field pumpkin, purification

Shigella spp. are Gram-negative intracellular pathogenic bacteria belonging to the family Enterobacteriaceae. The pathophysiological impact of the bacteria is highly related to the composition and structural variability of lipopolysaccharides. Serum sensitivity and biofilm forming ability are correlated with the length of these molecules, while bacteria with truncated lipopolysaccharides are more sensitive to hydrophobic antibiotics. Inhibitors of lipopolysaccharide biosynthesis have the potential to develop new antimicrobial agents or antibiotic adjuvants. Bacterial two-component systems enable bacteria to sense and to respond to the changes in different environmental conditions. This study focuses on the inhibition of the rfaD gene encoding the ADP-L-glycero-D-mannoheptose-6-epimerase, which is involved in the lipopolysaccharide biosynthesis. Although, there are some inhibitors presumed for bacterial two-component systems like Closantel, their impact on lipopolysaccharide biosynthesis has not been examined previously. The Shigella sonnei 4303 strain was involved in the experiments with known lipopolysaccharide structure. The effect of Closantel on lipopolysaccharide biosynthesis and the limitations of its use are presented.

Keywords: lipopolysaccharide, lipooligosaccharide, lipopolysaccharide biosynthesis, Closantel, Shigella sonnei

This study explores the covalent immobilization of three lipases (Lipase AK, from Pseudomonas fluorescens; Lipase PS, from Burkholderia cepacia; and CrL, from Candida rugosa) on four supports prepared by functionalization of mesoporous hollow silica microspheres (M540) with various bisepoxides as activating agents for production of novel lipase biocatalysts for enantiomer selective biotransformations of secondary alcohols. The influence of length, rigidity and hydrophobicity of the bisepoxide activating agents was investigated on the efficiency of immobilization and catalytic properties of the resulted twelve lipase biocatalysts. The hollow silica particles modified with the most beneficial bisepoxide activating agents resulted in novel biocatalysts capable for kinetic resolution of racemic 1-phenylethanol rac-1a and racemic octan-2-ol rac-1b with high activity and enantioselectivity.

Keywords: hollow silica microspheres, bisepoxide surface activation, kinetic resolution, lipase, immobilization

This study describes the development of a continuous-flow procedure for the enzymatic kinetic resolution of rac-1-phenylethan-1-ol with the clickable acylating agent 2,2,2-trifluoroethyl 2-(prop-2-yn-1-yl-oxy)acetate, which allows facile click reaction-based downstream, enabling the largescale chemo-enzymatic synthesis of both secondary alcohol stereoisomers with high enantiopurity. The influence of flow rate, acylating agent and substrate concentration upon the productivity of the packed-bed reactor was investigated. In addition to this, the performances of continuous-flow and batch reactors were compared.

Keywords: enzymatic kinetic resolution, secondary alcohol, lipase, continuous flow process, batch process, productivity

Cephalosporins, a large group of β-lactam antibiotics, contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C, and substitutions of chemical groups or modifications of 7-ACA side-chains resulting in varying pharmacologic properties and antimicrobial activities, development of useful antibiotic agents, also. Cephalosporin C obtained by fungus fermentation can be transformed to 7-ACA by two-step or one step enzymatically conversion process. The most important step in 7-ACA downstream process is represented by its separation from enzymatically produced reaction mixture. Among the used methods new separation techniques have been developed and applied to bioseparations, like reactive extraction and pertraction which have considerable potential.

Keywords: 7-aminocephalosporanic acid, cephalosporin C, enzymatic reaction, pertraction, reactive extraction

Apoptotic regulation has been implicated in many human diseases, including cancer, autoimmune disease, inflammation and neuro degradation. Mapping up critical apoptosis regulators is a strategy for the development of new therapies [1, 2]. Present work highlights optimization of heterologous expression conditions for the X-linked inhibitor of apoptosis protein (XIAP). Genes of target protein containing pGEX-4T vector was transformed in chemically competent E. coli Rosetta™(DE3)pLysS cells. The recombinant construct contained a glutathione S-transferase (GST) fusion partner, which assured the purification of the protein by affinity chromatography. In the next step we examined the growth dynamics of the expression culture in M9 minimal medium, meanwhile we also determined the appropriate time of induction. Following this we carried out the optimization of expression, examining the expression’s effectiveness under different conditions. On the basis of these fermentation experiments the target protein expression was the most prominent at 18 °C with 0.2 mM IPTG induction for 12 hours. During large scale fermentation experiments, we followed the optical density (OD), dry cell weight and substrate utilization. Finally, recombinant protein expression inhancement in the presence of 3% ethanol was successfully achieved in bioreactor. In this case the target protein was expressed in inclusion bodies, therefore solubilisation and refolding is necessary.

Keywords: XIAP, heterologous expression, optimization, bioreactor, ethanol

The phytochemical composition of apple, cranberry, pomegranate, carrot, and beetroot juices was determined in terms of carotenoids, anthocyanins, betalains and ascorbic acid concentration. Antioxidant potential of these beverages was also assessed using the colorimetric DPPH radical scavenging assay. The results showed that pasteurized and unpasteurized carrot and apple juices contain significant amount of β-carotene and cis-β- carotene. High total betalain content was observed in beetroot juice. Major anthocyanins detected in cranberry and pomegranate juices were glycosides of cyanidin, malvidin, and delphinidin. Notable ascorbic acid content and antioxidant potential were observed in unprocessed carrot juices, while beetroot juice exhibited the highest percentage of free radical inhibition.

Keywords: juices, ascorbic acid, carotenoids, anthocyanins, antioxidant activity

A comparative study for analysis of essential oils obtained from plants grown in Transylvania, plants belonging to Apiaceae family, was achieved. Five types of plants seeds from Coriandrum sativum L. (coriander), Anethum graveolens L. (dill), Pimpinella anisum L. (anise), Carum carvi L. (caraway) and Foeniculum vulgare L. (fennel) were hydrodistilled to yield essential oils. The chemical composition of the obtained essential oils was determined; also the antioxidant, antimicrobial and tyrosinase inhibitory activities were investigated. The major constituent of coriander essential oil were linalool (73%), for dill (52.7%) and caraway (67.55%) was carvone and for anise (95.2%) and fennel (89.64%) was trans-anethole. The anise seeds essential oils provide the highest antioxidant activity while the coriander essential was the lowest. The fennel essential oils proved to be a potent tyrosinase inhibitor. All studied essential oils exhibited considerable antimicrobial activity, but coriander essential oil proved to have a very high antibacterial activity on gram-negative bacteria E. Coli and Klebsiella pneumonia.

Keywords: essential oils, antioxidant activity, tyrosinase inhibitors, antimicrobial activity, GC/MS

The present paper was aimed to establish mathematical models useful to reduce the time required to discover the appropriate adsorption conditions of Congo Red (an intensively used organic dye) on an activated carbon prepared from cherry stones through calcination. To this purpose, various values of three parameters known as influencing the process, namely dye initial concentration (200 mg/L to 1000 mg/L), pH (2 to 12) and contact time (10 to 180 minutes) between the adsorbent and the adsorbate were variated. The recorded results of the adsorption process were used as data for Response Surface Methodology and Artificial Neural Network and several mathematical equations were generated. The conducted statistical analyses revealed that these equations can accurately express the Congo Red elimination from aqueous solutions. Moreover, the developed procedure is able to predict the process evolution in different conditions than those experimentally tested.

Keywords: Adsorption, Artificial Neural Network, cherry stone, Congo Red, mathematical modelling, Response Surface Methodology, water treatment

1,4-butanediol (BDO) is an important commodity molecule that is used as a platform chemical for the production of polybutylene terephthalate (PBT), elastic fibres (Spandex) and other materials. The homologous enzyme of E. coli, succinyl-CoA synthetase (sucCD) and the heterologous malonyl-CoA reductase from Chloroflexus aurantiacus (mcr) are key enzymes in a heterologous pathway leading to BDO production, which were introduced into a genome-engineered E. coli MG1655(DE3) ΔldhA, ΔpflB strain. Knowing that the expression of recombinant proteins and gene deletions can significantly influence cellular viability, the present study was carried out to investigate the impact of the two key enzyme expression on deletion strains, helping us to analyze the physiological changes of E. coli strains and providing directions for further optimizations in order to achieve satisfying target product yields.

Keywords: 1,4-butanediol, heterologous enzyme, Escherichia coli, metabolic engineering

A potential enantioselective access to 3-amino-7-aryl-8- azachromans is described. The reported multi-step synthesis involved the use of aspartic acid as the building block that could induce chirality. The azachroman scaffold was obtained via a key intramolecular inverse Diels- Alder cycloaddition between a 1,2,4-triazine and a judiciously functionalized alkyne. This class of compounds are hetero-isosteres of previously reported ligands of the histaminergic H3 receptor.

Keywords: 8-azachroman, inverse Diels-Alder cycloaddition, H3R ligands

In this work we describe the synthesis and characterization of new Mannich bases derived from 2-phenylaminothiazole, by applying the trimolecular condensation of thiazole aldehydes with aniline and acetone, catalyzed by lipase B from Candida antarctica. The target compounds were obtained with 68-76% yields, in mild and eco-friendly reaction conditions. The new heterocyclic Mannich bases were characterized by melting points, 1H NMR, 13C NMR and MS spectrometry.

Keywords: thiazole, Mannich base, lipase, multicomponent reactions

Compounds of type Ph2P(E)SeR [E = S, R = 2-MeC6H4 (1), 2-(Me2NCH2)C6H4 (2); E = Se, R = 2-(Me2NCH2)C6H4 (3)], were prepared and structurally characterized in solution by multinuclear NMR (1H, 31P, 77Se). The attempts to obtain the derivative Ph2P(S)SeC6H4(CH2NMePri)-2 (4) resulted in a complex mixture. The attempts to separate the components resulted in further decomposition and hydrolysis and the isolated crystals were investigated by single-crystal X-ray diffraction when proved to be the ammonium salt [MePriBnNH]+[Ph2P(S)O]¯ (Bn = benzyl) (4a).

Keywords: organophosphorus(V) compounds, organoselenium compounds; intramolecular coordination; solution behavior, solid state structure

Detection of bacterial volatile metabolites produced by human pathogenic bacteria is gaining continuous interest in both scientific and medical fields. Solid-phase microextraction (SPME) is a sampling technique that gained increasing attention in the last years due to its simplicity to implement and sensitivity. Volatile organic compounds (VOCs) released in the headspace over exudates infected with different bacteria were investigated in this work. GC-MS was involved for analysis. The above mentioned VOCs resulted from bacterial metabolism and the afferent processes that occur inside the biological samples. However, we identified 14 compounds emitted by the infecting pathogens, which can be assumed as bacterial markers.

Keywords: bacterial markers, exudates, headspace profiles, diagnostic tool

The objective of the study was to determine in vitro – in vivo correlations for Gliclazide 60 mg modified release tablets developed by Ranbaxy Laboratories Limited, now Sun Pharmaceutical Industries Limited, India, based on the data obtained in two bioequivalence clinical trials and in in vitro dissolution tests. Each clinical trial was designed as an open-label, randomized, single-dose, crossover study that consisted of two periods. The first bioequivalence study was performed under the fasting state of the subjects, while the second bioequivalence study was carried out under the fed state of the subjects. During each study period, venous blood samples were taken pre-dose and post-dose up to 96 hours. Afterwards, individual plasma profiles were obtained and mathematical deconvolution was applied to obtain the relative fraction absorbed of gliclazide. These data were correlated with the in vitro dissolution data obtained after performing dissolution tests in three different dissolution media, at pH 4.5, 6.8 and 7.2, with Gliclazide 60 mg modified release tablets (EvoluPharm, France). All calculation were performed by Phoenix WinNonlin® version 6.3. For each in vivo data set from the bioequivalence studies (under fasting and fed conditions), three level A in vitro-in vivo correlations were obtained for Gliclazide 60 mg modified release tablets, for in vitro tests performed at pH 4.5, 6.8, and 7.2. Good correlation coefficients were found for each established correlation (R2=0.98-0.99). In conclusion, six level A in vitro-in vivo correlations were obtained for Gliclazide 60 mg modified release tablets manufactured by Ranbaxy Laboratories Limited, now Sun Pharmaceutical Industries Limited, India.

Keywords: gliclazide, in vitro-in vivo correlations, clinical trial, healthy Caucasian subjects

The aim of the study was to propose an improved HPLC/UV method for captopril impurity profiling based on forced degradation studies. Material and methods: the HPLC-UV analyses were performed on a Luna C18 column at 50 0C by using a mobile phase consisting of phosphoric acid 15 mM and acetonitrile at 210 nm. Results and discussions: an HPLC method with superior characteristics to one described in the European Pharmacopoeia for captopril impurities profiling was proposed. The main degradation product of captopril was captopril disulfide (Impurity A), the concentration of which increases in all the conditions of forced degradation, except thermal degradation. UV light has led to the highest number of unknown impurities, but the oxidative degradation presented the highest rate of degradation (>88%). A total of 15 unknown impurities have been observed with significant area (>0.50%), who were not initially present in the captopril sample. Conclusions: The proposed HPLC method can be successfully applied in pharmaceutical analysis laboratories as a stability indicating method of captopril, allowing the separation of official impurities A, B, C, D and E and those formed under forced degradation conditions.

Keywords: captopril, impurities, stability indicating method, degradation

Quantitative structure-retention relationships (QSRR) approach was used to model chromatographic lipophilicity of sixteen proteinogenic amino acids using molecular descriptors computed with DRAGON and ALCHEMY software packages. Modeling was performed applying multiple linear regression (MLR) coupled with genetic algorithms (GA) methodology (MLR-GA). The most important descriptors, highly significant in the predictive models of amino acids lipophilicity (RM0), were related to atomic polarizabilities (MATS3p; Ap; H1p), atomic van der Waals volume (MATS3v), Sanderson electronegativity (RDF070e) and Randic molecular profiles (DP11; DP12) calculated with Dragon software. The internal statistical evaluation procedure highlighted some appropriate models for the chromatographic lipophilicity prediction. Moreover, the statistical parameters of regression in order to evaluate the relationship between experimental and predicted values, in case of the test set (four amino acids), revealed three statistically valid models (model A, E and F) that can be successfully used in lipophilicity prediction of amino acids.

Keywords: chromatographic lipophilicity, amino acids, multiple linear regression, genetic algorithm, molecular descriptors, modeling, prediction

The purpose of this study was the development and validation of an LC-MS/MS method, for the determination of diclofenac from human plasma. The sample workup involved a simple protein precipitation procedure. A core/shell type analytical column (50×2,1 mm, 2.6 Å) was used with C18 stationary phase. The mobile phase consisting of 52.5% acetonitrile and 47.5% water provided good peak shape, accuracy and precision (stable ionization). The mass spectrometer was operated in negative electrospray ionization mode for analyte and internal standard. The following parameters were evaluated for validation purpose: Selectivity, sensitivity, matrix effect, anticoagulant effect, linearity, precision and accuracy, recovery, short and long term analyte/IS stability in solvent/matrix and carryover. The validated calibration range was 3.9-1194 ng/ml. The correlation coefficient R2 was at least 0.999 in all validation batches. The validated method has been successfully used for the evaluation of bioequivalence of a generic diclofenac potassium formulation of 12.5 mg strength.

Keywords: diclofenac, NSAID, method validation, bioequivalence trial, LCMS/MS

Nitrite content was determined in fresh meat of chicken and pork and in different processed meat products such as sausages, salami, ham and pate with declared and not declared nitrite addition. The spectrophotometric method based on diazo-coupling reaction was used. Under the optimum working conditions, the method showed a limit of detection of 0.4 mg kg-1 and a limit of quantification of 1.2 mg kg-1. The method proved to be accurate with nitrite recovery of 98±14% from spiked samples. In the analyzed samples the nitrite content was in the range 1.1±0.1 to 26.4±2.5 mg Kg-1, values that are below the maximum admitted level of nitrite in meat products (150 mg Kg-1). The lowest nitrite concentration was found in chicken meat products (between 1.1±0.2 and 1.8±0.1 mg kg-1) while in salami, ham and sausages products the content was high (26.4±2.5; 19.0±0.7 and 13.4±1.1 respectively). The health risk exposure parameters were between 4% and 94 % and between 2% and 50% from established Acceptable Daily Intake (ADI of 0.07 mg/kg body weight/day) in case of adults and children respectively. Higher values correspond to pork meat products as salami, sausages and ham. The risk exposure to nitrite estimated for occasional consumption of meat products proved to be much more informative than the nitrite concentration. For these products consumption should be limited to 150-300 g per week. Noncarcinogenic health effects, evaluated based on Target Hazard Quotient (THQ) were revealed for the investigated meat products by occasional consuming of 3 serving/week.

Keywords: nitrite, meat product, UV/Vis spectrophotometry, risk exposure assessment

By using the dielectric spectroscopy technique in the extremely low frequency (1–200Hz) range, dielectric loss tgδ was determined on both Aspergillus niger cultures in different growth stages and autoclaved biomass compared to the Czapex-Dox culture medium with sterile sucrose. Experimental results have shown that living and sterile matter samples have different dielectric characteristics. In sterile probes, the evolutionary functions of tgδ depending on frequency are continuous, specific to α- relaxation governed by DC electrical conductivity. In living matter samples, the function of dielectric losses tgδ, depending on frequency, show several discontinuities that indicate the existence of some processes in which, at those frequencies, the number of charge carriers from investigated living matter is changing. It has also been found that the package of active processes differs depending on the growth stage of Aspergillus niger.

Keywords: ELF electromagnetic fields, dielectric loss, relaxation dynamics, α-relaxation, fungi

The purpose of this study was the development and validation of an LC-MS/MS method, for the determination of solifenacin from human plasma. The sample workup involved a simple protein precipitation procedure. A core/shell type analytical column (50×2,1 mm, 2.6 Å) was used with C18 stationary phase. The mobile phase consisting of 65% acetonitril and 35% water provided good peak shape, accuracy and precision. The mass spectrometer was operated in positive electrospray ionization mode for analyte and internal standard. The following parameters were evaluated for validation purpose: Selectivity, sensitivity, matrix effect, anticoagulant effect, linearity, precision and accuracy, recovery, short and long term analyte/IS stability in solvent/matrix and carryover. The validated calibration range was 0.71-71.28 ng/ml. The correlation coefficient R2 was at least 0.99 in all validation batches. The validated method has been successfully used for the evaluation of bioequivalence of generic solifenacin 10 mg formulations.

Keywords: solifenacin, muscarinic antagonist, method validation, bioequivalence trial, LC-MS/MS

The present study aimed to elucidate and describe the basic pharmacokinetics of dapagliflozin after a single dose oral administration of a 10 mg immediate release tablet developed by Sun Pharmaceutical Industries Limited, India. Ten competing models were created in order to analyze the experimental data obtained from the 71 subjects who were enrolled and finalized two bioequivalence clinical trials, under fasting and fed state. The studies took place at the Clinical Pharmacology and Pharmacokinetics Department of Terapia S.A. Considering the Akaike index value for a rational model discrimination, model number 8 (M8) was found to be the best that fits the experimental data. The representative pharmacokinetic model involves zero order absorption kinetics with a lag time of approximately 0.3 hours, first order systemic metabolism and elimination and two-compartmental distribution. Furthermore, by using M8, the most important pharmacokinetic parameters of dapagliflozin were determined. All calculations were performed by Phoenix WinNonlin® version 6.3 (Certara, USA).

Keywords: dapagliflozin, compartmental pharmacokinetic analysis, subjects, bioequivalence clinical trials