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Vol 23, No 12 (2020)


Detection of a SARS-CoV2 RNA using multiplex reverse transcription loop-mediated isothermal amplification with melting curve analysis

Oscorbin I.P., Shevelev G.Y., Pronyaeva K.A., Stepanov A.A., Pyshny D.V., Filipenko M.L.


Relevance. Ongoing COVID-19 pandemic caused by the SARS-CoV2 coronavirus urges the need for diagnostic tests to detect the RNA of the SARS-CoV2 virus. The most common method, RT-PCR, allows to obtain the test results within 1.5-2 hours. However, the lack of capacity of diagnostic laboratories necessitates a developing of more rapid testing methods. Purpose of the study. The development of a method for a detection of SARS-CoV2 RNA using multiplex reverse transcription loopmediated isothermal amplification with melting curve analysis Material and methods. The constructed plasmids with a fragment of the SARS-CoV2 genome and MS2 phage, fragments of the SARS-CoV2 genomic RNA and MS2 phage were used as standard samples. Clinical samples (nasopharyngeal swabs) were obtained from patients CNMT of ICBFM SB RAS. RNA was isolated using a «RIBO-prep» kit (Central research Institute of Epidemiology (Moscow; Russia)). LAMP was performed in a CFX96 thermocycler (Bio-Rad; USA). Analytical characteristics of LAMP were evaluated using dilutions of standard samples. The clinical sensitivity and specificity of multiplex LAMP were evaluated by testing clinical samples simultaneously with LAMP and RT-PCR. Results. Primers were selected and conditions were optimized for a LAMP-based detection of SARS-CoV2 RNA and MS2 phage RNA, the latter served as an internal control of RNA isolation and amplification. Multiplexing was based on a melting curves analysis of amplification products. The limit of detection of the multiplex LAMP was 20 copies of SARS-CoV2 RNA per reaction. The concordance with real-time RT-PCR of the 40 clinical samples testing results was 92%. Conclusion. We have developed a prototype of a multiplex LAMP-based test system for a detecting SARS-CoV2 coronavirus RNA. The developed approach can be used as an alternative to RT-PCR in diagnostic practice for saving of machine and personnel time.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):3-10
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Standardization and value of the antioxidant activity of Onopordum acanthium herb

Garsiya E.R., Pozdnyakov D.I., Shamilov A.A., Logvinenko L.A., Konovalov D.A.


Relevance. The raw material of folk medicinal plants must be guality controlled according standardization technigue for using in the official medicine practice including preventing agents. Standardization technigue of aerial part of folk medicinal plant Onopordum acan-thium L. and the antioxidant activity of extracts were developed. Aim. Development and validation of guantitative determination method of total amount of phenols in the aerial part of Onopordum acanthium L, establishment antioxidant activity of extracts. Material and methods. Spectrophotometry was used as standardization method of Onopordum acanthium raw material. Antioxidant activity was carried out in vitro by DPPH, superoxide, hydroxyl and nitrosyl radicals tests. Results. The optimal conditions of isolation phenols are grinding size 1 mm, extracting solvent - ethanol 70% at the ration 1:50, time of extraction - 60 minutes. The extraction is heating. The guantitative analysis was carried out spectrophotometry using wavelength 329 nm and estimated as gram of caffeic acid at 100 g of air-dried material. The sum of phenols at the herb of O. acanthium was at 1,44% to 2,71% (in various places of natural growth). Method was validated by linearity, precision and accuracy. The highest antioxidant activity was established to water-alcohol extract (ethanol 70%). Conclusions. The lowest amount of phenols in the Onopordum acanthium aerial part may be 1,2 g of caffeic acid per 100 g of air-dried raw material. The antioxidant activity is higher for 70% alcohol extract with highest amount of phenols from the herb of Onopordum acanthium in tests with DPPH, superoxide, hydroxyl and nitrosyl radicals, which was comparable to caffeic acid
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):11-17
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Synthesis of molecular imprinted polymer based on acrylic acid derivatives for sample preparation of pyrrolidinophenone derivatives from biological fluids

Voronin A.V., Synbulatov I.V.


The study objective was to obtain a sorbent, which is a molecularly imprinted polymer based on acrylic acid derivatives, selective to compounds of the pyrrolidinophenone derivatives. Material and methods. Sorbents were obtained by the radical polymerization method of methacrylic acid, acrylamide, N,N'-methylene-bis-acrylamide, diallylamine in the presence of a template - phenylpiracetam. Amount of acrylamide and N, N'-methylene-bis-acrylamide used was varied. For the obtained sorbents, the degree of swelling in water and isopropanol, the imprinting factor, the cross-reaction coefficient, and the phenylpiracetam recovery from water solutions were determined. Results. The study of the degree of swelling of the obtained sorbents showed that the volumetric swelling in water and organic solvents increases with an increase in the content of acrylamide and a decrease in N,N'-methylene-bis-acrylamide. The volumetric swelling in water of all the obtained sorbents is lower than in isopropanol, which makes it possible to use them for analytes sorption from water and desorption. The value of the phenylpiracetam imprinting factor for the sorbent sample containing 56% acrylamide was 47% higher than for the sample containing 44% acrylamide. The low value of the imprinting factor of all sorbent samples in the study of cinnarizine and amitriptyline indicates that the obtained sorbents contain "molecular imprints" of the template, phenylpiracetam. The above sorbent samples are capable of recognizing phenylpiracetam in binary mixtures, which confirms the presence of selective binding sites. Phenylpiracetam recovery from water solutions using a sorbent sample containing 56% acrylamide was 89±17%. Structure analysis of this sorbent sample by FTIR revealed the availability of pronounced peaks at 1451, 1660, 2934, 3198, 3343 crml, which indicates the presence of carboxy-modified polyacrylamide matrix of the sorbent. Conclusion. A molecularly imprinted sorbent based on acrylic acid derivatives was obtained, which is stable in water and isopropanol. The produced sorbent exhibits a high level of selectivity to pyrrolidinophenone derivatives, which has been proven in an experiment with a model compound phenylpiracetam. This polymer sorbent can be used for sample preparation of biological fluids in the study for pyrrolidinophenone derivatives.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):18-23
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Component composition of essential oils and antioxidant activity of Hyssopus officinalis L. cultivars introduced in the mountainous conditions of Dagestan

Kuramagomedov M.K., Aliev A.M., Islamova F.I., Mamalieva M.M., Radjabov G.K., Musaev A.M.


Relevance. Currently, there is an acute problem of strengthening the raw material base of essential oil and spicy-aromatic plants. For the Republic of Dagestan, the culture of Hyssopus officinalis L. is new and unexplored in mountain conditions. We are screening crops that can be used to restore terraced farming, a traditional mountainous industry that is now in deep decline and can be used in the pharmaceutical and food industries. Purpose of the research. Comparative study of the component composition and antioxidant activity in varieties Hyssopus officinalis L. introduced in the mountainous conditions of Dagestan and their comprehensive assessment as possible crops for terraced farming. Material and methods. The objects of study are four varieties: «Accord», «Goluboy», «Dachny lekar», «Otradny». Essential oils were obtained by steam distillation. The composition of essential oils was determined by gas chromatography with a mass spectrometric detector. To determine antioxidants, the amperometric method was used on a «Tsvet Yauza 01-AA». Results. It has been established that the content of essential oil of the studied varieties ranges from 0,4 to 0,6 %. A high content in all varieties of pinocampheon was noted, as well as a relatively high content of pinocarvon in the essential oil of the «Otradnaya» variety. The major components beta-trans-ocimene and alpha-elemol are present only in the «Dachny lekar» essential oil. Components such as myrtenol and pinocarvone are present in the two varieties of essential oils «Accord» and «Otradnaya». Antioxidant activity in varieties varies within 31,7-49,5 mg/g. Conclusions. All varieties belong to the pinocampheon chemotype. The significant content of major components in the four types of essential oils can be considered as their potential resource for the production of essential oils of various uses. The obtained data on antioxidants should be taken into account when evaluating varieties as a source of antioxidants. Plants are resistant to return frosts and tolerate drought well in mountain-valley and mid-mountainous Dagestan.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):24-30
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Development and pharmacokinetic study of the original dosage form - gliclazide suspension

Storozhenko S.E., Kutyakov V.A., Stepanova E.F., Veselova O.F.


Relevance: one of the most common forms of diabetes mellitus (DM) is type 2 diabetes mellitus. A personalized approach to diabetes therapy presupposes adeguate glycemie control and the availability of original drugs with various pharmacokinetic parameters, incl. bioavailability. One of these dosage forms is a suspension that meets a number of good manufacturing practice (GMP) reguirements and has several advantages over other dosage forms. Purpose of the work: to study the bioavailability of Gliclazidum from a suspension in comparison with a tablet dosage form. Material and methods: the study of the bioavailability of Gliclazidum from the suspension and tablets was carried out on male Vistar rats weighing 190-200 grams. The animals were injected with the developed suspension of Gliclazidum and the reference drug - tablets "Diabeton MV" ("ServierRus", Russia, active ingredient - Gliclazidum). Form of administration of drugs - intragastrically at the rate of 60 mg / kg of body weight of animals. Blood sampling was carried out at intervals from 2 to 24 hours after drug administration. Extraction of Gliclazidum from blood plasma was carried out with dichloromethane at a pH of 9.5, the extract was dried, dissolved in 100 pi of methanol, and the solution was analyzed by liguid chromatography. The Gliclazidum content was calculated using the internal standard method. A calibration graph was preliminarily constructed, where a "blank" (not containing gliclazide and anecaine - internal standard) blood plasma was used. The study was carried out on a liguid microcolumn chromatograph "Milichrom - A02" with a UV spectrophotometric detector. Results: it was found that the concentration of Gliclazidum after 2, 4 hours, as well as its residual concentration in the blood plasma of rats 12 and 24 hours after administration of the suspension is higher than after administration of tablets. The data obtained indicate a higher bioavailability of the drug from the suspension compared to the tableted dosage form. Conclusions: a higher bioavailability of Gliclazidum from a suspension than from tablets has been experimentally established, which confirms the feasibility of its medical use.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):31-35
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Analysis of metabolic changes in liver mitochondria and red blood cells in essential hypercholesterolemia in rats

Mikashinovich Z.I., Semenets I.A., Romashenko A.V.


Relevance. One of the main causes of increased mortality is vascular atherosclerosis against the background of hypercholesterolemia. The aim of the study was to analyze the biochemical changes occurring in liver cells and red blood cells in the simulation of essential hypercholesterolemia in rats. Material and methods. In liver mitochondrial homogenates and blood red blood cells in the control and experimental groups, indicators of the main energy cycles characterizing substrate-enzyme processes (pyruvic acid, lactate, cytochrome oxidase and succinate dehydrogenase), as well as antioxidant protection (reduced glutathione, glutathione reductase and glutathione peroxidase) were determined. Results. The changes recorded in the liver may reflect adaptive shifts associated with the accumulation of pyruvate as an additional substrate for mitochondrial oxidation, while in red blood cells, the accumulation of lactate contained with a decrease in the level of pyruvate indicates the formation of hypoxia. The decline of glutathione level in the liver and in erythrocytes there is a tendency to increase, which can be linked to increased transport of reduced glutathione from liver to blood. This pattern reflects the increasing role of red blood cell reduced glutathione both in regulating gas transport function and protecting cell membranes from the destructive effects of O2. Conclusion. Inhibition of glutathione peroxidase activity in essential hypercholesterolemia in both liver cells and red blood cells contributes to a violation of the balance of oxidized glutathione/reduced glutathione, which accelerates atherosclerotic manifestations.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):36-41
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Immunomodulating properties of the complex plant supplement «Gipeliv»

Ubeeva E.A., Khobrakova V.B., Nikolaev S.M., Ubeeva I.P., Khitrikheev V.E.


A necessity in finding new hepatoprotective drugs and supplements is dictated by the growing demand: increasingly widespread liver pathologies tending to chronic forms being caused by viruses, toxic agents including some medication. The aim of the work was to determine immunomodulating properties of the complex plant supplement consisting of Hypecoum erectus L, Hedysarum dauricum, Glycyrhhiza uralensis Fisch., Calendula officinalis and Scutellaria baicalensis. The effect of the studied plant supplement was measured by assessement of cellular, humoral and macrophageal components of immune response in the mouse model of azathioprine induced immunodeficiency. The experiment was carried out on male FI line mice (CBAxC57BI/6). Immunosuppressive agent azathioprine was administered to the control group of animals 50 mg/kg orally once daily during 5 days. The experimental group received same doses of azathioprine and the plant supplement 100 mg/kg once daily for 14 days. The state of the cellular component of the immune response was evaluated by following the standard method of the delayed-type hypersensitivity reaction (DTH) assessment, while the macrophage component was studied in the reaction of phagocytosis of peritoneal macrophages, and the assessment of humoral immunity was measured by the number of antibody-forming cells.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):42-45
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Changes in the level of antioxidants in the leaves of Rosa majalis and Fragaria viridis after acaricidal treatment of forest with deltamethrin

Chigrinski E.A., Gerunova L.K., Konvay V.D., Shorin N.V., Zolin P.P.


Relevance. The phytotoxic effects of pesticides are characterized by a decrease in biomass, a slowdown in the growth of the aerial part of plants or their root system, as well as a change in biochemical processes in cells. The effect of synthetic pyrethroids on plants of the Rosaceae family, which includes medicinal plants, has not been deeply studied, and therefore it is of great interest to determine the level of both antioxidant accumulation and other biologically active substances in various parts of plants of this family when exposed to pesticides. Objective. To establish the effect of the synthetic pyrethroid deltamethrin on the level of antioxidants in the leaves of wild rose (Rosa majalis) and green strawberry (Fragaria viridis) during acaricidal forest treatment. Material and methods. R. majalis and F. viridis were the objects for studying. Samples for the study were collected from an experimental forest site with an area of 1 ha at 7, 30 and 60 days after acaricidal treatment of the area with deltamethrin. Alongside, samples were collected at the background site. In the leaves of R. majalis and F. viridis, collected from both sites, the content of ascorbic acid, tocopherol, glutathione, as well as the activity of catalase, superoxide dismutase, and glutathione reductase were determined under laboratory conditions. Results. Deltamethrin causes a decrease in the level of non-enzymatic antioxidants and activation of antioxidant defense enzymes in R. majalis in the early stages after acaricidal treatment of the area. In the late periods of observation, all the studied parameters, with the exception of glutathione reductase activity, reached the background level. In the early stages of the effect of deltameth rin on F. viridis, a decrease in the content of non-enzymatic antioxidants in the leaves was observed, and in a later period, inhibition of the activity of antioxidant enzymes was observed against the background of a persisting deficiency of non-enzymatic antioxidants. Conclusion. It was found that acaricidal forest treatment with deltamethrin activates antioxidant enzymes and decreases the content of ascorbate, tocopherol and glutathione in the leaves of R. majalis and F. viridis. These changes are more pronounced in F. viridis.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):46-51
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Studying morphogenic potential of annual clary (Salvia viridis L.) in vitro

Zotova E.P., Cherednichenko M.Y.


Salvia viridis L. is widely cultivated as an ornamental plant, but it has medicinal value, including antioxidant, antibacterial and antifungal properties. The presence of flavonoids, caffeic acid derivatives and phenylethanoids may enhance its potential antioxidant properties. Plasmic seeds of S. viridis (PLAZMAS Ltd.) were used in the research. To obtain aseptic plant material, the seeds were sterilized with 5% sodium hypochlorite for 5, 10, and 15 minutes, then washed twice with distilled water. The seeds were placed on to phytohormone-free Murashige and Skoog (MS) nutrient medium. Petiole, leaf and stem expiants were cultivated on MS nutrient medium without NH4NO3 with the addition of various phytohormones and growth regulators: indolyl-3-acetic acid (IAA), a-naphthaleneacetic acid (NAA), indolyl-3-butyric acid (IBA), 6-benzylaminopurine (BAP); a phytohormone-free nutrient medium was used as a control. Based on the results of experiments, 5% sodium hypochlorite solution with holding time of 5 minutes can be recommended for surface sterilization of annual clary seeds. This mode provides the high yield of aseptic plants. The study of the morphogenic potential of S. viridis aseptic plants makes it possible to increase the multiplication factor of this valuable plant. It has been shown that stem and petiole expiants should be used for the induction of callusogenesis by cultivating expiants on MS nutrient medium with or without phytohormones and growth regulators. A high freguency of rhizogenesis can be obtained on petiole and stem expiants on MS nutrient medium with the addition of 1 mg/L IAA or NAA.
Problems of Biological Medical and Pharmaceutical Chemistry. 2020;23(12):52-55
pages 52-55 views

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