Vol. 19 No. 3(75) (2021)
Published:
2021-10-20
Original Researches
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Modern chemical disinfectants and antiseptics. Part I
Aim. To generalize and systematize information on the properties of modern chemical disinfectants and antiseptic agents (DA and AA).
Results and discussion. The review provides generalized and systematized information on the properties of modern chemical DA and AA – alkylating reagents, aldehydes, amides, amidines, bisguanidines, dyes, halogenated reagents, halogens and their complexes, 2-nitrofuran derivatives. The classification of DA and AA by their chemical structure was carried out. The activity spectra, possible application ways and forms of DA and AA were given. Their toxicity and impact on the environment were described as well.
Conclusions. On the basis of the analysis carried out it was shown that aldehydes, halogen-active compounds and halogen-containing complexes are modern effective DA and AA with a wide spectrum of biocidal action. Amides, amidines and bisguanidines are characterized by a narrow spectrum of activity. Dyes and 2-nitrofuran derivatives are old-fashioned antiseptics. -
The synthesis, antimicrobial activity and docking studies of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin- 4(3H)-ones with acetamide and 1,2,4-oxadiazol-5-ylmethyl substituents
Aim. To synthesize, study the antimicrobial activity and suggest antimicrobial activity mechanism for the
novel derivatives of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4(3H)-one.
Results and discussion. As the result of the targeted modification of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]-pyrimidin-4(3H)-one in position 3 with acetamide and 1,2,4-oxadiazol-5-ylmethyl substituents, the compounds, which demonstrated better antimicrobial activity in the agar well diffusion assay than the reference drug Streptomycin, were obtained. To elucidate the mechanism of action of the novel compounds, the docking studies were con-
ducted to the active site of the 16S subunit of ribosomal RNA, the proven target for aminoglycoside antibiotics, as well as tRNA (Guanine37-N1)-methyltransferase (TrmD), which inhibitors were considered as a new potential class of antibiotics.
Experimental part. By the interaction of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4(3H)-one with a series of N-arylchloroacetamides and 3-aryl-5-(chloromethyl)-1,2,4-oxadiazoles in DMF in the presence of K2CO3 the target compounds were obtained. The antimicrobial activity was assessed by the agar well diffusion method. The concentration of microbial cells was determined by the McFarland standard; the value was 107 cells in 1 mL of the media. The 18 – 24 hour culture of microorganisms was used for tests. For the bacteria cultivation,
Müller-Hinton agar was used, Sabouraud agar was applied for C. albicans cultivation. The compounds were tested as the DMSO solution with the concentration of 100 µg/mL; the volume of the solution was 0.3 mL, the same volume was used for Streptomycin (the concentration 30 µg/mL). The docking studies were performed using Autodock Vina. Crystallographic data for the complexes of Streptomycin with the 16S subunit of ribosomal RNA
(1NTB) and its active site, as well as for tRNA (Guanine37-N1)-methyltransferase (EC 2.1.1.228; TrmD) (5ZHN) and its active site were obtained from the Protein Data Bank.
Conclusions. It has been determined that 2-[6-(1H-benzimidazol-2-yl)-5-methyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl]-N-[4-(ethoxy)phenyl]acetamide, which is the most active as an antimicrobial agent among the compounds tested, also shows the best binding activity towards the active site of tRNA (guanine37-N1)-methyltransferase. -
The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves
Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.
Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton.
Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.
Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising. -
Determination of catechins in green tea leaves by HPLC compared to spectrophotometry
Aim. To study the qualitative composition, the quantitative content of catechins in green tea leaves and compare the data obtained with those evaluated by spectrophotometry.
Materials and methods. Green tea leaves used for the analysis were collected in Anhui Province, China. The extract for the HPLC analysis was obtained by the maceration method with 60 % ethanol twice in the raw material/extractant ratio of 1 : 20. In the case of the spectrophotometric analysis, green tea leaves were extracted with 70 % ethanol twice by the maceration method in the raw material/extractant ratio of 1 : 20. The analysis of the extract from green tea leaves was performed by high performance liquid chromatography using a Prominence LC-20 Shimadzu chromatographic system (Japan) with a SPD-20AV spectrophotometric detector, an Agilent Technologies Microsorb-MV-150 column (reversed phase, C18 modified silica gel, length – 150 mm, diameter – 4.6 mm, particles size – 5 μm). Substances in the extract were identified by comparing the retention time and the spectral characteristics of the test substances with the same characteristics of the reference standards. Spectrophotometric measurements were carried out using a UV-1000 single beam spectrophotometer (China) with the pair of S90-309Q quartz square cells.
Results and discussion. Using high performance liquid chromatography 5 catechins were identified.
Among them epigallocatechin-3-O-gallate (10.85 %) predominated, while catechin (0.61 %) had the lowest concentration. The total amount of catechins in green tea leaves was 30.56 and 24.79 % by HPLC and spectrophotometry, respectively. The F- and t-tests showed that there was no significant difference between the results of HPLC and spectrophotometry.
Conclusions. The qualitative composition and the quantitative content of catechins have been determined in the extract from green tea leaves by high performance liquid chromatography and spectrophotometry. Both HPLC and spectrophotometric methods can be used to determine the total catechin content in green tea leaves. The high content of catechins makes the extract promising for further study and creation of new herbal medicinal products and dietary supplements. The results obtained will be used for standardization of green tea leaves and for future pharmacological research of its extract. -
The determination of the cholinesterase activity using 3,3′,5,5′-tetramethylbenzidine as an indicator
Aim. To develop a new method, which has a good reproducibility of the experimental results, is fast, cheap and provides safe working conditions during the analysis, in order to determine the activity of cholinesterase.
Experimental part. The light absorption of the test and control samples was measured using a CPhC-3-01 photoelectric photometer (420 nm, l = 3 cm). The reaction rate was characterized by the value of the optical density of the solution in 10 min (the fixed time method). Measurements were performed at +37 °C, the temperature of the reaction mixture was maintained by thermostatіng in water, the pH of the solutions was monitored potentiometrically using a glass electrode. The determination was repeated five times with each solution of a certain concentration of the enzyme. According to the average values obtained, the calibration graph of the specific activity of the enzyme (in international units – activity unit (AU/mg) – kmol/min to 1 mg of the substance) on the optical density of the solution was constructed. Using the mean value of five measurements of the optical density of the test solution the specific activity of the enzyme (U) was found by the calibration graph.
Results and discussion. The essence of the method is the photometrical measurement of the rate of the enzymatic hydrolysis of acetylcholine in a buffer medium using 3,3′,5,5′-tetramethylbenzidine (TMB). The enzymatic hydrolysis reaction of the substrate was performed at pH 8.3, and in 10 min after the start the rate of enzymatic hydrolysis of acetylcholine was measured. The linear dependence of the optical density on the specific activity of the enzyme (U) was observed in the range of 3.5 – 28 AU/mg (activity unit/mg). The activity of the enzyme, according to the average results of 5 measurements, was 27.9 AU/mg. The declared activity the enzyme in accordance with the quality certificate was 28 AU/mg. The limit of quantification was 0.2 AU/mg. Metrological characteristics of the method were as follows: RSD = 1.8 % (n = 5; P = 0.95), accuracy – 0.45 %. These values indicate that the method proposed for determining the activity of cholinesterase is characterized by high sensitivity, reliability and reproducibility of the results. At the same time, it was proven that there was no systematic error in determining the activity of cholinesterase by the method developed.
Conclusions. As a result of the research conducted a new method for determining the activity of the cholinesterase enzyme has been developed; it is characterized by high sensitivity, reliability and reproducibility of the results, and also provides safe working conditions during the analysis. -
The identification and the quantitative determination of loratadine by the HPLC method
Aim. To develop the unified method of the HPLC analysis of loratadine, which can allow obtaining reliable and reproducible results of the studies of pharmaceuticals and biological matrices for monitoring the treatment effectiveness.
Materials and methods. The HPLC analysis was performed on a “Milichrome A-02” microcolumn liquid chromatograph under the following conditions: a reversed-phase variant, 2 × 75 mm column filled with a non-polar sorbent Prontosil 120-5 C18 AQ, 5 μm; the mobile phase in the mode of a linear gradient – from eluent А (5 % of acetonitrile and 95 % of a buffer solution) to eluent B (100 % of acetonitrile) for 40 min. The flow rate of the mobile phase was 100 μL/min; the injection volume was 4 μL. The multichannel detection of the substance
was carried out using an UV-detector at 210, 220, 230, 240, 250, 260, 280 and 300 nm; the optimal value of the column temperature was 37 – 40 °С, and the pump pressure was 2.8 – 3.2 MPa.
Results and discussion. As a result of the studies performed, the retention parameters of loratadine and spectral relationships were determined using the unified HPLC method. This made it possible to include the results obtained in the database for the identification of antihistamines in the therapeutic monitoring of the treatment with an individual drug or in the complex treatment of allergic reactions. The development of the quantitative determination
of loratadine by HPLC on model solutions using various concentrations of the drug was carried out. The content of loratadine was determined by the equation S = 1.14 × 10-3C – 0.50 × 10-4; the correlation coefficient was 0.9998. It was found that the relative standard deviation RSD did not exceed 0.93 % when analyzing loratadine in the model solutions by HPLC.
Conclusions. The identification and the quantitative determination of loratadine by the unified HPLC method have been conducted. The method allows obtaining reliable and reproducible research results. The results of the studies can be recommended for implementation in the practice of forensic bureaus, toxicological centers, and clinical laboratories. -
The component composition of the essential oil from Laurus nobilis L. unripe fruits harvested in Ukraine
Aim. To study the qualitative composition and the quantitative content of the essential oil components from Laurus nobilis L. unripe fruits.
Results and discussion. In the essential oil from Laurus nobilis L. unripe fruits 31 compounds were determined; among them 28 substances were identified. A high content was characteristic for spatulenol (1947.1 mg/kg) and betulenol (925.3 mg/kg).
Experimental part. The raw material for obtaining the essential oil (unripe fruits) of Laurus nobilis L. was harvested in November 2017 in Alushta and the village of Rybalskoe, Crimea. The component composition of the essential oil of the unripe fruits from Laurus nobilis L. was studied and the constituents of the essential oil were identified by chromato-mass spectrometry using an Agilent Technology 6890N chromatograph. The component composition of the essential oil was revealed by comparing the results obtained with data from the NIST 02 mass spectrum library (more than 174.000 substances).
Conclusions. The component composition of the essential oil from Laurus nobilis L. unripe fruits harvested in Ukraine has been studied. Taking into account the set of the biologically active compounds found in the essential oil of Laurus nobilis L. it can be argued that further pharmacognostic studies of fruits of this plant as the medicinal raw material with the predicted pharmacological activity (e.g., antimicrobial and skin cleansing) are promising.
