Problems of Biological Medical and Pharmaceutical Chemistry
ISSN (print): 1560-9596, ISSN (online): 2587-7313 Founders: All-Russian Scientific Research Institute of Medicinal and Aromatic Plants, Ministry of Science and Higher Education of the Russian Federation Editor-in-Chief: Sidelnikov Nikolay Ivanovich, Academician of the Russian Academy of Sciences, Doctor of Agricultural Sciences Frequency / Access: 12 issues per year / Subscription Included in: White List (4th level), Higher Attestation Commission List, RISC
Pharmacognostic characteristics of Artemisiae altaiensis herba
Introduction. Plants of the genus Artemisia L. are a valuable source of phenolic and terpene compounds, which accounts for their widespread use in both traditional and conventional medicine. The State Pharmacopoeia of the Russian Federation (SP RF) currently includes only Artemisiae absinthii herba, highlighting the importance of studying closely related species to expand the range of herbal medicines. Artemisia altaiensis is an underexplored endemic species of the Altai-Sayan Mountain system and Mongolian Altai. A comprehensive pharmacognostic analysis of its aerial parts is necessary to better understand its potential. Objective – to conduct a pharmacognostic analysis of Artemisiae altaiensis herba. Material and Methods: The aerial part of Artemisia altaiensis was collected in the Republic of Tuva in August 2018. The authenticity and quality of the raw material were evaluated following the methodologies outlined in the State Pharmacopoeia of the Russian Federation (SP). Anatomical and diagnostic characteristics were determined after treating the samples with clearing reagents using a Lomo μVizo-10 microscope. The moisture content, total ash, acid-insoluble ash (in 10% HCl solution), and total extractable substances were quantified gravimetrically in five replicates. Qualitative analysis was performed according to standard protocols. The quantitative determination of flavonoids was carried out using a UV spectrophotometer PE 5400 at a wavelength of 405 nm. Essential oils were extracted by hydrodistillation following method 2 of the State Pharmacopoeia, and their component composition was analyzed by gas chromatography-mass spectrometry (GC-MS) using an Agilent Packard HP 6890 chromatograph. Results. Morphological and anatomical diagnostic characteristics of the raw material were established. Qualitative tests identified the presence of flavonoids, essential and fatty oils, tannins, coumarins, and polysaccharides. The quality indicators of the raw material were as follows: moisture content (5,78±0,18%), total ash (5,41±0,19%), acid-insoluble ash (5,00±0,25%), and the content of extractive substances obtained with 70% ethanol (37,40±0,46%). The presence of impurities was also assessed. The quantitative flavonoid content, expressed as rutin equivalents, was determined for various morphological parts: aerial parts (1,10±0,03%), inflorescences (1,53±0,09%), leaves (1,71±0,06%), and stems (0,44±0,03%). The essential oil content of A. altaiensis was found to be 0,20%. Major components included 9-Epi-caryophyllene, α-eudesmol, caryophyllene oxide, β-selinene, acephyllic acid. Conclusions. The authenticity and quality parameters of Artemisia altaiensis herb have been determined. Analysis of the essential oil composition revealed the presence of compounds with pronounced anti-inflammatory, antibacterial, and antioxidant properties, substantiating the potential use of A. altaiensis in medicine.
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Component composition of the essential oil of Umbellularia californica (Hook. & Arn.) Nutt.)
Introduction. In order to search for new potential sources of biologically active substances, research was carried out for the first time on the essential oil of Californian Umbellularia (Umbellularia californica (Hook. & Arn.) Nutt.), obtained from raw materials grown in the Russian Federation. Aim – the article presents the results of studying the quantitative and qualitative composition of the essential oil of Umbellularia californica (Hook. & Arn.) Nutt.), obtained in a humid (Black Sea coast of the Caucasus) and moderately dry subtropical climate of the Mediterranean type (Southern Coast of the Crimea), as well as a comparative analysis of its indicators with those from other regions of this species. Material and methods. The procurement of plant materials (fresh leaves) to obtain essential oil was carried out in October 2021). The essential oil was obtained by hydrodistillation using Ginsberg apparatus. The qualitative composition of essential oils was determined using a Chromatek-Crystal 5000.2 chromatograph equipped with a mass spectrometric detector. Results. It was found that in the conditions of a dry, moderately warm Mediterranean climate of the Southern Coast of the Crimea, the mass fraction of essential oil in fresh leaves is 1.2% (2.7% in terms of absolutely dry weight), in samples from humid subtropics of the Black Sea coast of the Caucasus – 5.1% (13.0% in terms of absolutely dry weight). A comparative analysis of the component composition of Umbellularia essential oil from various growing sites allowed us to conclude that, regardless of the region of cultivation, method and duration of distillation, its major components are umbellulone (36.7–41.0%) and 1.8-cineol (12.8–22.0%). In the conditions of the Southern Coast of the Crimea, terpinene-4-ol (11.99%) and thymol (11.05%) are present in essential oil; methylevgenol (9.9%) and sabinene (7.67%) on the Black Sea coast of the Caucasus; methylevgenol (8.4%) and thymol (7.8%) in Italy (Genoa); in natural conditions (USA, Oregon, Toledo) – sabine (10.2%). Conclusion. The conducted research allows us to consider Umbellularia californica as a promising source of valuable essential oil with a consistently high content of umbellulone and 1,8-cineole for cultivation in the conditions of the Black Sea coast of the Caucasus.
Qualitative and quantitative composition of protective phenolic compounds in aqueous and aqueous-ethanol extract of brown algae of the genus Cystoseira
Introduction. Herbal medicines are used in medical practice to treat a wide range of diseases. Marine organisms, especially brown algae, have attracted great interest worldwide due to their potential use in various fields of medicine. Algae are considered to be a rich storehouse of bioactive compounds such as sulfated polysaccharides, carotenoids, phytosterols, phycobiliproteins, phloroglucin, etc. The presence of polysaccharides such as alginate, laminarin and sulfated fucoidan, etc., makes them more significant compared to other algae. Recently, there has been an increasing trend among researchers to use algae as a source of natural antioxidants. The aim of the study was to evaluate the qualitative and quantitative composition of phenolic compounds in the aqueous and aqueous-ethanol extract of brown algae of the genus Cystoseira barbata, collected on the shelf of the eastern coast of Crimea in the summer, as well as to evaluate their antiradical properties. Material and methods. To obtain an extract of phenolic compounds, a sample of frozen brown algae of the genus Cystoseira barbata, collected in July 2022 in the bays of the eastern coast of Crimea, was dried to a constant mass at a temperature of 100–105 0C. Phenolic compounds from a sample of dry raw materials were extracted by the method of digitization. Water and 70% ethyl alcohol were used as solvents. A qualitative and quantitative analysis of the extracts was performed, as well as an assessment of their antiradical properties. Results. The presence of tannins was assumed in the obtained extracts by qualitative analysis. By the methods UV- and IR-spectroscopy have confirmed this assumption. It has been established that water extracts contain a higher amount of phenolic compounds, however, water-ethanol extracts have a higher antiradical activity. The resulting discrepancy can be explained by the presence in the alcohol extract of compounds belonging to the class of xanthines, which may also have antiradical properties. It is possible that the antioxidant effect of alcohol extracts have a synergistic effect, however, this assumption requires additional research. Conclusion. Thus, our data open up broad prospects for further study of brown algae of the genus Cystoseira barbata, growing on the Black Sea coast, as a source of biologically active compounds of the phenolic and xanthine series. These groups of compounds are of interest to the pharmaceutical industry as protectors of slowing down the aging process, preventing age-related diseases, and having a protective effect on the ischemic zone.
Development of a thermo-sensitive in situ nasal gel with nanoparticles of poly(lactic-co-glycolic) acid copolymer containing favipiravir
Introduction. Favipiravir is a selective inhibitor of viral RNA polymerase with a broad spectrum of antiviral activity; however, its low aqueous solubility results in reduced oral bioavailability. Therefore, the development of new delivery systems, such as thermosensitive nasal in situ gels, is necessary. The purpose of the work. To develop and characterize thermosensitive in situ gels based on P407 and P188 for intranasal delivery. Material and methods. Favipiravir-loaded PLGA nanoparticles were prepared using nanoprecipitation. Particle diameter was deter- mined using dynamic light scattering (DLS) at 25 °C. Thermosensitive gels were prepared using the cold method with varying P407 concentrations and varying the P407/P188 ratio. Rheological properties were measured using a SmartPave 102 rheometer at 10–40 °C and a heating rate of 0.05 °C/sec. In vitro release studies were conducted using a DHC-6T transdermal diffusion tester in phosphate buffer, pH 6.4, at 37 °C, with samples analyzed at six time points using UV spectrophotometry. Results. The average diameter of favipiravir-loaded PLGA nanoparticles was 168.14±3.49 nm, a suitable size for intranasal delivery via the nose-to-brain mechanism. In situ temperature-sensitive gel studies revealed that as the P407 concentration increased from 18 to 25% by weight, the Tsol-gel decreased from 29.04±0.63 °C to 21.84±0.19 °C. The addition of P188 at an 8/2 ratio (P407/P188) significantly increased the T in the sol-gel by 6.64–8.38 °C compared to systems without P188. The favipiravir release profile from the API suspension was characterized by rapid release (46% in 5 min, 72% in 10 min); from PLGA nanoparticles, by delayed release (17– 29% in 5–10 min, 94% in 120 min); and from the in situ gel with nanoparticles, by the most prolonged release (3–9% in 5–10 min, 65% in 120 min). Conclusion. An intranasal delivery system consisting of PLGA nanoparticles loaded with favipiravir in a thermosensitive in situ gel ma- trix based on P407 and P188 was developed. A dual release control mechanism (polymer degradation and delayed diffusion from the gel matrix) provides a pronounced prolonged effect.
Development of the composition of the dry nasal spray vinpocetine and the technology of its production
Introduction. Vinpocetine has neuroprotective properties and is used to improve cognitive function; however, poor solubility and low bioavailability may limit its use. The development of a dry nasal spray vinpocetine with increased solubility is a topical issue. Aim of the study was to develop the composition of a prototype dry nasal spray of vinpocetine and the technology for its production. Material and Methods. The prototypes contained chitosan and synthesized poly-β-cyclodextrin. The formation of host-guest inclusion complexes by co-dissolution and co-attrition methods increased vinpocetine solubility. IR spectroscopy and X-ray diffraction analysis were used to confirm the formation of inclusion complexes and evaluate the sample properties. Spray drying was the final step in the formation of the dry nasal spray. The stability and mass (volume) uniformity of doses taken from a multi-dose package, dosing in a dry powder dispenser, and in vitro permeability in a transdermal diffusion tester were studied for the lead formulation. Results. Comparative studies of the developed samples with different vinpocetine:polycyclodextrin ratios were conducted. No significant differences in inclusion complex formation were demonstrated by the two methods. Therefore, the co-dissolution method was chosen as the preferred method for producing dry nasal spray prototype samples, as it is technologically simpler. In vitro permeation results showed that most of the vinpocetine dissolves and penetrates the membrane within 30 minutes. Dosage uniformity and stability of the developed prototypes for 12 months were demonstrated. Conclusions. The experimental studies demonstrate the feasibility of using powder particles containing the vinpocetine-polycyclodextrin inclusion complex and chitosan, obtained by spray drying, as prototypes for a dry nasal spray for vinpocetine.
Synthesis of L-selenocystin, N,N'-diacetyl-L-selenocystin and their investigation as antioxidants and glutathione peroxidase mimics
Introduction. Cysteine and its derivatives have unique biological functions; acetylcysteine and carbocysteine are registered and marketed drugs. Selenium cysteine derivatives have to be a great challenge due to their vital role in natural redox catalysis and their use in dietary supplements. The adequate space for chemical modification creates a complex interests to manipulate their properties for pharmacological and nutritional purposes. The aim of this study was to synthesize and investigate the antioxidant and glutathione peroxidase mimic properties of L-selenocystine and N,N'-diacetyl-L-selenocystine. Material and Methods. Chemical synthesis of selenocystine from serine, acetylation of the amino group of selenocystine, to get acetylselenocystine. Coulometric assays of the antioxidant capacity and activity of cysteine and its derivatives. The reduction reactions of the diselenide with mercaptoethanol and N-acetylcysteine were studied spectrophotometrically. The glutathione peroxidase mimic properties of L-selenocysteine were modeled by bromine oxidation and sodium borohydride reduction. Results. Sulfur-containing antioxidants have higher antioxidant capacity than selenium-containing. The antioxidant capacity in acidic solutions decreases in the following order: Cystine > L-selenocystine > N-acetylcysteine > N,N'-diacetyl-L-selenocystine > Methionine. An experimental coulometric conditions were developed for antioxidant activity assays. The antioxidant activity of N-acetylcysteine was higher than that of N,N'-diacetyl-L-selenocystine. Diselenide exhibits the glutathione peroxidase mimic properties: 1) accelerates the thiol oxidation and 2) is capable for reversible oxidation. In the red-ox model of selenocystine bromine oxidation and sodium borohydride reduction the 93% of substance were regenerated. Conclusions. Diselenide bond of selenocysteine can be used as a chromophore for the spectrophotometric assays. N-acetylcysteine is weaker reductant for the diselenide bond than mercaptoethanol.
Development and validation of a chromato-mass-spectrometric method for quantitative determination of apigenin, apigetrin and luteolin in mice plasma
Introduction. Currently, the practical interest of researchers is aimed at studying the biomedical potential of secondary plant metabolites in order to create new drugs. Flavonoids are a large class of natural phenolic compounds with a wide range of biological activity. Particular attention is paid to the ability of some flavones (apigenin and luteolin, their glycoside apigetrin) to suppress the growth of malignant tumors. However, low concentrations of these polyphenols in plant matrices do not allow reliable assessment of pharmacokinetics in the human body. In this regard, an urgent bioanalytical task is the development of accurate and reproducible methods using high-tech hybrid methods of analysis. Purpose of the study. Development and validation of chromato-mass-spectrometric methodology of the quantitative determination of apigenin, apietrin andlLutheololin in the blood plasma of mice. Material and methods. To develop the methodology of quantitatively determined flavones, a highly effective liquid chromatograph Agilent Technologies 1260 Infinity II and the mass spectrometer Ab Sciex Qtrap 3200 MD was used. The chromatographic separation was carried out on the analytical column Agilent Infinitylab Poroshell 120 EC-C18 with a gradient mobile phase containing 0.1 % of ant and acetonitril. For detecting, analytes and the internal standard warfarin were ionized by electrical exposure in the mode of negative ions using multiple reactions monitoring. The sampling of plasma of the blood of mice was precipitating with methanol. Results. The optimal conditions of the convertible-phase chromatographic separation of apigenin, apigerin, lutheololin and varfarin with times of retention from 5,3 to 7,8 minutes were selected. Each compound in optimal conditions of electrical ionization is protoned with the formation of the composition of the composition [М–Н]- and fragmentation on two characteristic ion-products. The methodology of simultaneous quantitative HPLC-MS/MS determination of flavones in the plasma of mice, characterized by a linearity in a wide range of concentrations and a low limit of quantitative determination of 1.0 ng/ml, values of the degree of extraction of analyts in the range of 90,8-100,4 %. The validation characteristics of the methodology satisfy the relevant regulatory requirements of selectivity, the influence of the matrix effect, intraulinary accuracy and precision. Conclusion. The results of validation tests indicate the suitability of the developed express and rather accurate bioanalytic methodology of the quantitative determination of the three structurally related flavons in the joint presence of mice in the blood plasma. The metrological characteristics of the methodology allow you to use it to conduct the analytical part of pharmacokinetic studies of drugs containing both individual flavonoids and plant extracts.
One-carbon metabolism prognostic markers in the development of acute myocardial infarction in patients after COVID-19
Introduction. The SARS-CoV-2 virus affects the cardiovascular system, having long-term consequences, and leads to an increased risk of developing CVD diseases. To prevent the development of complications, it is important to search for predictors of cardiovascular pathology. Purpose of the study – to evaluate the prognostic significance of homocysteine, folic acid, and genetic polymorphisms of folate cycle and endothelial synthase genes in the development of acute myocardial infarction (AMI) in patients after a new coronavirus infection. Material and methods. A case-control study, the study group consisted of patients after COVID-19, confirmed by PCR, of moderate or severe form, for a period of 6 to 24 months, the main group with AMI, the control group without AMI. All patients underwent studies of serum homocysteine and folic acid levels and molecular genetic studies (MTHFR 677 C>T, MTHFR 1298 A>C, MTR 2756 A>G, MTRR 66 A>G, NOS3 -786 T>C, NOS3 894 G>T). Results. In patients after COVID-19, a violation of one-carbon metabolism due to hyperhomocysteinemia was found, as well as a high risk of AMI in middle-aged patients with an increase in homocysteine levels in the blood of more than 14.3 mmol/l and carriage of the NOS3 -786 gene. Conclusion. An imbalance of one-carbon metabolism markers and the presence of polymorphism of endothelial synthase genes contributes to the development of acute myocardial infarction after COVID-19 in middle-aged patients.
Study of HIF-1α, HIF-2α and VEGF-A dynamics in plasma as potential markers of endothelial dysfunction and acute respiratory distress syndrome in patients with severe COVID-19 pneumonia
Introduction. Impaired adaptation to hypoxia in COVID-19 pneumonia may lead to endothelial dysfunction and acute respiratory distress syndrome (ARDS). In this regard, the prognostic value of determining plasma levels of HIF-1α, HIF-2α, and VEGF-A has been actively studied, but their significance in relation to the development of ARDS is poorly understood. Aim of the study – to evaluate the dynamics of HIF-1α, HIF-2α and VEGF-A in plasma of patients with COVID-19 pneumonia as potential markers of endothelial dysfunction and ARDS development. Material and Methods. HIF-1α, HIF-2α and VEGF-A levels were studied in plasma at the time of admission and on the 7th day of treatment in 90 patients (47 women, 43 men) aged 31 to 86 years (median 65 years) with different severity of COVID-19 pneumonia (moderately severe course – 35, severe course – 28, extremely severe course – 27). The amount of HIF-1α, HIF-2α and VEGF-A was determined in blood plasma using ELISA kits. Results. Patients with extremely severe disease had significantly lower plasma HIF-1α levels on admission than patients with moderate severity, while VEGF-A levels were significantly higher. These differences were also reflected in clinical outcomes, as HIF-1α level on admission was positively correlated with the risk of developing ARDS (r=0.483, p<0.01), while VEGF-A level was negatively correlated with ROX index (r=–0.339, p<0.05) and associated with a lower risk of developing ARDS (r=-0.378, p<0.05). Conclusions. Patients with severe COVID-19 pneumonia have elevated levels of VEGF-A upon admission, indicating the development of endothelial dysfunction. Plasma HIF-1α and VEGF-A levels on admission, but not on day 7, are associated with the risk of ARDS in COVID-19 patients. Lower HIF-1α and higher VEGF-A at admission indicate increased risk. Evaluation of baseline and dynamics of markers of adaptation to hypoxia and development of endothelial dysfunction is important for predicting COVID-19 outcomes.
Diagnostic characteristics of a test system for the detection of viral hepatitis B by real-time PCR from the results of analysis of clinical samples
Introduction. PCR method is important in the clinical diagnostics of hepatitis B virus (HBV), especially at low viral load, while monitoring the dynamics of treatment and when identifying the occult form of infection. There are strict requirements for the diagnostic sensitivity and specificity of PCR test systems used in clinical practice. The aim of the study was to determine the diagnostic characteristics of the developed by us test system for PCR diagnosis of hepatitis B virus (HBV) on 837 human plasma samples. Material and methods. 465 human plasma samples obtained from people with a confirmed diagnosis of HBV infection were examined, as well as 372 blood plasma samples obtained from people with a confirmed absence of HBV in blood plasma. All samples were obtained from an accredited laboratory. The commercial “RealBest HBV PCR kit (form 2)” (JSC Vector-Best, Russia) was used as a comparison kit. The RealBest Deltamag HBV/HCV/HIV kit (JSC Vector-Best, Russia) was used to isolate nucleic acids on Auto-Pure96 automatic station (Allsheng, China). To evaluate the effectiveness of nucleic acid isolation, internal control sample was also added to the human blood plasma sample. Results. Developed by us test system was compared with the "RealBest HCV PCR" test system. From the analysis of clinical samples of human blood plasma using both test systems, the diagnostic characteristics of the developed by us test system were determined as 96.39% diagnostic sensitivity and 98.33% diagnostic specificity. Conclusions. The statistical analysis of Bland-Altman showed a high degree of similarity of sensitivity of both test systems used. The developed by us test system can be recommended for use in the clinical diagnosis of HBV.
Biological activity of the purified extract from the herb Dracocephalum moldavica L.
Introduction. The search for plants that can become sources of BAS for the creation of new highly effective and safe medicines is one of the promising areas of pharmaceutical science. Moldovan snakehead (Dracocephalum moldavica L.) has long been used as an anti-inflammatory, wound healing, expectorant and sedative, as well as an essential oil and aromatic herb. A dry purified extract from Moldovan snakehead grass with a rosemary acid content of about 56.6% was obtained at VILAR State Medical University. The aim of the study was to conduct a primary screening of the biological activity of an extract from Moldovan snakehead grass in in vitro and in vivo experiments. Materials and methods. The primary screening of the biological activity of the extract was studied using specific enzyme biotest systems in vitro based on glutathione reductase, catalase, and inducible NO synthase. The capillary-strengthening effect of the extract was studied according to the "Method for determining the reactivity of skin capillaries to inflammatory stimuli" based on a model of a local inflammatory reaction (xylene inflammation) in rats. Results. The results of the conducted studies indicate that when introduced into the incubation medium, the extract had a direct (120% higher relative to the control) effect on the rate of the glutathione reductase reaction, that is, it contributed to an increase in the antioxidant protection of thiol enzymes involved in muscle contraction through the natural antioxidant glutathione. At the same time, the extract under study showed anti-inflammatory activity, reducing the rate of reaction catalyzed by inducible NO synthase. In vivo experiments showed that the studied extract of Moldovan snakehead grass had a significantly pronounced capillary-strengthening effect in the experimental model of xylene inflammation, significantly increasing the time of petechiae appearance by 37%, and the time of distinct petechiae staining by 21% compared with the control. Conclusions. As a result of in vitro experiments, it was shown that the activity of the dry extract of Moldavian snakehead grass is manifested through the binding of BAS to the enzymes lutathione reductase and inducible NO synthase, which suggests possible mechanisms of antioxidant and anti-inflammatory effects of this plant. An experimental model of xylene inflammation using laboratory animals has shown that at a dose of 200 mg/kg, the extract has a pronounced capillary-strengthening effect.
Wound-healing effect of pectin nanogel with encapsulated sea buckthorn (Hippophae rhamnoides L.) oil in in vivo experiments
Introduction. One of the important tasks of medicine is the healing of burn wounds, since the process of renewal of damaged tissues takes a long time and can be accompanied by complications. The use of new technologies and drugs, such as nanogels with encapsulated oils, has excellent prospects for accelerating wound healing and improving tissue regeneration. The use of pectin nanogel with sea buckthorn oil (Hippophae rhamnoides L.) can significantly affect both the effectiveness of burn wound therapy and the acceleration of the regeneration process. Purpose of the study – evaluation and characterization of morphophysiological changes in the skin and the functional state of fibroblasts in the burn wound area when using pectin nanogel with encapsulated sea buckthorn oil. Material and methods. In this study, a polysaccharide nanogel based on low-methoxylated apple pectin and apricot gum with encapsulated sea buckthorn oil (Hippophae rhamnoides L.) was developed – hereinafter referred to as "Gel OM". Comparative studies were conducted on the effectiveness of burn wound healing using the nanogel "Gel OM" with the liquid patch "Plast" on 32 same-sex white outbred rats. The experimental animals were divided into two groups – 16 individuals in each. Group A is the main group using "Gel OM" (pectin nanogel with sea buckthorn oil). Group B is the control group using the liquid patch "Plast". The main 2 groups, depending on the observation time, were additionally subdivided by days of observation into subgroups: A1, A2, A3, A4 and B1, B2, B3, B4. The experiment was carried out with subsequent morphological analysis of tissues taken from the burn wound area. Results. The study used tissues taken from the wound area, which were subjected to various methods of processing and staining for subsequent analysis. In both groups, microscopic examination of tissue sections showed that wound healing on the 3rd and 7th days occurred at the same rate. However, in group B3 on the 14th day, the re-epithelialization process was more pronounced than in group A3. On the 21st day, in group A4, uneven regeneration of the epithelium with areas of hyperproliferation was observed, while in group B4 wound healing occurred faster and more completely. Conclusions. The results of this work showed that in groups A and B, healing occurs according to the type of reparative regeneration with complete restoration of the histological structure of the epidermis and dermis. However, in group B (using the nanogel "Gel OM"), the healing process and formation of granulation tissue is accelerated in relation to group A (using the liquid patch "Plast"). Thus, the use of the polysaccharide nanogel form with encapsulated sea buckthorn oil ("Gel OM") in the main group showed greater efficiency in restoring burn wounds in animals compared to the liquid patch "Plast" in the comparative group. "Gel OM" demonstrated good results and this allows us to recommend this developed product for use in medicine to restore the histological structure of the skin, to accelerate the regeneration of burn wounds, especially at later stages of healing.
Effect of the complex plant extract on the state of the immune system of the body in experimental immunodeficiency
The purpose of the study was to evaluate the immunomodulatory effect of a complex plant remedy in experimental immunosuppression caused by azathioprine. Material and methods. The object of the study is a dry extract of the following composition: Serratula centauroides (L.), Bergenia crassifolia (L.) Fritsch., Rosa davurica Pall., Inula helenium L., Echinacea purpurea (L.) Moench.). Experiments were carried out on F1 (СВАхС57Вl/6) mice. Immune deficiency was simulated by intragastric administration of azathioprine in the dose 50 mg/kg. The first and second experimental groups administered the dry polyextract (100 mg/kg) and a comparative drug «Echinacea» (200 mg/kg) respectively intragastrically once a day for 14 days against the background of azathioprine immune suppression. The intact group administered the purified water according to the analogous scheme. The effect of the tested remedy on the cellular immunity was evaluated in the reaction of the delayed-type hypersensitivity (DTH), the humoral immunity was estimated by the number of antibody-forming cells (AFC), the functional activity of macrophages was assessed in the phagocytosis reaction of peritoneal macrophages against colloidal ink particles. Results. The complex plant extract increased the DTH-index by 1.4 times and the value of absolute and relative numbers of AFC by 1.4 and 1.6 times, phagocytic index by 1.8 times, as compared with the control group; it was comparable to the action of «Echinacea». Conclusion. The complex plant extract has an immunomodulatory effect on cellular and humoral immunity as well as the functional activity of macrophages in azathioprine immunosuppression.
Quantitative determination of a new sidnonymine derivative in blood plasma and organs of mice using HPLC-MS/MS
Introduction. Sydnonymines are a class of heterocyclic chemical compounds with a wide range of biological activity and promising in terms of the search and development of new drugs. One of these compounds is the candidate molecule BBP2023, N-(ethoxycarbonyl)-3-(4-methylhexane-2-yl)sydnonymine. It has been determined that BBP2023 accumulates in the brain tissues of laboratory animals and causes a cerebral vasodilating effect, which makes it possible to consider this compound as a promising drug used to treat cerebral circulatory disorders. Goal – development and validation of a bioanalytical method for the determination of a new sidnonymine derivative in various tissues of laboratory mice with a view to further application in experimental pharmacokinetic research. Material and methods. Quantitation of BBP2023 in blood plasma and organ homogenates of mice was performed by HPLC-MS/MS. The technique of precipitation of proteins with methanol was used as the basis for biomaterial sample preparation. Chromatographic separation was performed with a mixture of deionized water and acetonitrile in a gradient mode on a Zorbax Eclipse XDB-C18 4,6×50 mm, 5 µm column. Detection was performed in the MRM mode. Results. As a result of the experiment, a technique for sample preparation of biological material of mice was developed, optimal conditions for chromatography and detection of the studied compound were selected. The bioanalytical technique has been validated according to the following parameters: selectivity, matrix effect, recovery rate, carry-over, linearity of the calibration range, lower limit of quantitative determination, intra- and inter-run accuracy and precision, dilution factor, as well as stability of the analyte and the internal standard at the analysis stages. Conclusion. As a result of the experiment, a method was developed for the quantitative determination of a new derivative of sydnonymine BBP2023 in blood plasma and internal organs of mice. The analytical range of the method ranged from 5 to 5,000 ng/ml for blood plasma and from 25 to 25,000 ng/g for liver, kidneys, heart, lungs, brain, spleen, skeletal muscles and adipose tissue. The bioanalytical method fully meets the validation requirements and can be used to perform the analytical part of an experimental study of the pharmacokinetics of BBP2023, including an assessment of the distribution of the studied compound across organs and tissues.
Effect of Akkermansia muciniphila-based probiotic on small intestinal morphology and gene expression and behavioral characteristics of mice under induced systemic inflammation
Introduction. The microbiome plays an important role in the normal functioning of the body, an imbalance in its composition leads to various diseases. Currently, many studies are being conducted on the bacterium Akkermansia muciniphila, which is a new generation probiotic that has a beneficial effect on the composition of the microbiome. However, the mechanism of the effect of the bacterium on inflammatory processes occurring in the body and induced by various drugs has not been fully studied. The aim of the study was to determine the effect of A. muciniphila on serum biochemical parameters, as well as behavioral and physiological characteristics of mice during lipopolysaccharide (LPS)-induced systemic inflammation. Material and methods. The following methods were used in the study: physiological test "Open field", biochemical analysis of blood serum using the automatic analyzer "VitaLine 200", histological examination of the small intestine was carried out using hematoxylin and eosin staining, gene expression was assessed by RT-PCR combined with reverse transcription. Statistical processing was performed in the R environment (Version 4.1.1) and Statistica 12, using the Spearman correlation coefficient, the Kruskal–Wallis test and the Bonferroni correction, one-way variance test (ANOVA) using the Tukey post-hoc test. Results. A. muciniphila inhibited the inflammatory process induced by LPS injections and, as a result, led to an increase in research activity. The restoration of the small intestine structure was observed. Also, against the background of the probiotic effect, normalization of the biochemical parameters of the blood serum was revealed. Conclusion. With the combined administration of LPS and A. muciniphila, antioxidant protection (GCLC) and intestinal barrier function (ZO-1) were enhanced, thus, the probiotic demonstrates therapeutic potential in the course of the inflammatory process.
Phenolic precursors as regulators of the polyphenols accumulation in the tea plant callus cultures
Introduction. Plant tissue cultures are potential producers of biologically active plant metabolites, which include various phenolic compounds that can be used to maintain human health. In most cases, their accumulation is lower than in the initial explants, which requires a search for factors and influences to intensify this process. In this case, it is very promising to use the precursors of their biosynthesis as potential “regulators" of the formation of various metabolites. The aim of the study was to study the effect of L-phenylalanine (FA, 3 mM), trans-cinnamic acid (CC, 1 mM) and naringenin (NG, 0.5 mM), as components of various stages of phenolic metabolism, on the accumulation of various classes of phenolic compounds, including phenylpropanoids and flavanols, in callus cultures of the tea plant (Camellia sinensis L.): a strain of "Kimyn" and "Local population". Material and methods: The object of the study was callus cultures of tea plants of leaf origin grown for 7 days on a liquid nutrient medium of Heller basic composition (control) or enriched with L-phenylalanine (3 mM), trans-cinnamic acid (1 mM) or naringenin (0.5 mM). The morphophysiological parameters of the cullus (color, density, water content), the amount of polyphenols and flavans in them were analyzed. Results. According to the data obtained, the action of NG led to a significant increase in the accumulation of phenylpropanoids and flavanols in two strains of tea culture. Whereas FA contributed to a sharp increase in the content of phenylpropanoids only, which is more pronounced in the "Kimyn" strain. Thus, the most effective "regulators" of the accumulation of phenolic compounds are FA and NG. At the same time, the trends in the accumulation of various polyphenols were similar for both cultures, however, in the case of the "Wormwood" strain, the response was more pronounced. Conclusion. The study demonstrates the significant biotechnological potential of phenolic precursors as regulators of the accumulation of polyphenols in the callus cultures of the tea plant. The effect of their action depends on both the compound used and the strain of the tea plant. The results obtained open up prospects for the development of effective biotechnological approaches to the regulation of the accumulation of pharmacologically valuable secondary metabolites of a phenolic nature in in vitro cultures of higher plants.
Secondary metabolites and biotechnology of key lime (Citrus aurantiifolia (Christm.) Swingle)
Citrus aurantiifolia (key lime or mexican lime) belongs to the family Rutaceae, members of which are highly valued worldwide for their flavor, valuable nutrients, and bioactive compounds (BACs). Due to the biological activity of its secondary metabolites (SMs), lime and its components are used in traditional medicine for treating joint diseases, headaches, coughs, cardiovascular disorders, and hemorrhoids. The SMs of lime include alkaloids, carotenoids, coumarins, essential oils (including isoprenoids), and phenolic compounds (such as flavones, flavonoids, and phenolic acids). The juice, extracts, essential oil, and SMs of lime are widely used in the food and cosmetic industries, medicine, agriculture, and bionanotechnology as flavor enhancers, fragrances, preservatives, antioxidants, antimicrobial agents, herbicides, insecticides, and substances for metal recovery from precursor compounds during nanoparticle synthesis. Lime extracts have been noted for their ability to inhibit the growth of cancer cells and even induce apoptosis. The propagation of key lime using traditional methods is often inefficient and time-consuming, which hinders the rapid and large-scale production of BACs. The application of biotechnological methods allows for the optimization of the propagation process, the production of virus-free plant material, and the enhancement of SM synthesis. Biotechnological methods of lime propagation include the creation of «artificial seeds», somatic embryogenesis, and clonal micropropagation. The accumulation of target metabolites can be induced by modifying the composition of the nutrient medium, specifically through the application of phytohormones and the addition of NaCl at various concentrations. A promising approach in genetic engineering is Agrobacterium-mediated transformation. Although lime exhibits low susceptibility to infection by Agrobacterium strains, significant improvements in transformation efficiency have been achieved through protocol modifications and the use of antioxidants. Electrofusion of protoplasts is an effective method for obtaining interspecific and intergeneric lime hybrids. Thus, hybrids of Sudachi and Lime as well as Lime and Feroniella have been successfully produced. Lime extracts, due to the presence of stabilizing and modifying substances, are actively used in the green synthesis of nanoparticles. Nanoparticles of silver, gold, zinc oxide, copper oxide, and tin oxide have been synthesized from extracts of various plant parts.
Study of cultivation conditions of avar carnation (Dianthus awaricus Khar.) in vitro
Introduction. Dianthus awaricus Khar. is an endemic species of Dagestan flora, belonging to the group of white-flowered species and grown in experimental plots of the Mountain Botanical Garden of the DFRC RAS. This species is promising not only for ornamental gardening, but also due to its potential due to the presence of phenolic compounds and fatty oils in the seeds. The purpose of the study is to evaluate the cultivation conditions and the possibility of clonal reproduction of Avar carnation in vitro. Materials and methods. The experimental part of the work was carried out in the research laboratory of plant physiology and biotechnology named after Professor A.G. Yusufov at the biological faculty of the Federal State Budgetary Educational Institution of Higher Education "Dagestan State University". The objects of the study were the seed material of Dianthus awaricus, represented by the samples "Tsudakhar", "Gunib" and "Agvali". Using standard biotechnological approaches, various types of explants (nodal, apical and hypocotyl) were cultivated on Murashige-Skoog (MS) nutrient media supplemented with phytohormones: indole-3-butyric acid (IMA) and benzylaminopurine (BAP). Results. The combination of 1 mg/l BAP and 0.1 mg/l IMC showed the highest efficiency for nodal and apical explants of all the studied samples. The analysis of variance confirmed the significant effect of the type of explants on most of the studied traits (p < 0.05), so the factors "explants" and "phytohormones" had a statistically significant effect on the growth traits. The correlation analysis demonstrated the presence of strong positive relationships between the morphological parameters (height, number of roots, leaves and shoots). A high degree of correlation was established between the number of roots and the number of leaves (up to r = 0.99). Conclusions. The study of the in vitro cultivation conditions of Dianthus awaricus was conducted for the first time, which demonstrated the possibility of effective application of the tissue culture method for its propagation. Optimal conditions for explant cultivation for several introduced samples of Dianthus awaricus were proposed, allowing to significantly increase the reproduction rate and preserve the genetic potential of the endemic. The optimal nutrient media for propagation are combinations of phytohormones 2.5 mg/l BAP and 0.5 mg/l, as well as 1 mg/l BAP and 0.1 mg/l IMC (nodal and apical explants), IMC at a concentration of 0.5 mg/l (hypocotyl explants).