Problems of Biological Medical and Pharmaceutical Chemistry

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.