Vol 26, No 3 (2023)

Relevance. In the last 10 years, there has been an active study of the system of conjugation of the metabolism of a macroorganism and its microbiome. The microbiotic conversion of tryptophan into biologically active signaling molecules is a potential regulatory mechanism by which the intestinal microbiota can change the metabolism of both intestinal cells and the entire macroorganism. However, the content of tryptophan metabolism metabolites in patients with metabolically healthy (MHO) and metabolically unhealthy obesity (MUHO), as well as the relationship of these metabolites with adipokines and myokines, has not yet been studied.

Purpose of the study. To study the content of metabolites of tryptophan metabolism in the blood serum of obese patients and to evaluate the relationship between the content of adipokines and myokines and the content of tryptophan metabolism metabolites of bacterial and non-bacterial origin in the blood serum of patients with MSO and MNSO.

Material and methods. 266 patients were examined, including 138 healthy non-obese volunteers and 128 obese patients, of whom two subgroups were formed: 30 patients with MHO and 41 patients with MUHO. Metabolite concentrations in blood and feces were determined using high performance liquid chromatography. Quantitative analysis of adipokines and myokines was performed by multiplex enzyme immunoassay. The content of metabolites of tryptophan metabolism in blood serum was assessed by high performance liquid chromatography with mass spectrometric detection.

Results. For patients with obesity, an increase in the serum concentration of kynurenine, kynurenic and quinoline acids, indole-3-lactate, indole-3-butyrate and indole-3-acetate was established. Patients with MHO and MUHO were statistically significantly different only in terms of serum tryptamine concentration. Regardless of the presence/absence of metabolic disorders in obese patients, it has been established that xanthurenic and quinoline acids are interrelated with the concentration of myostatin in the blood serum. At the same time, for patients with MHO, it was shown that the serum concentration of the dominant catabolite of the indole pathway, indole-3-acetate, is interconnected with the content of insulin and leptin in the blood serum. Whereas in patients with MUHO, leptinemia is consistent with a high concentration of anthranilic acid in the blood serum, and hyperinsulinemia, on the contrary, is associated with a low concentration of indole-3-propionate in the blood serum.

Relevance. Mesenchymal stem-like stromal cells (MSCs) represent a promising material for the therapy of restoration or regeneration of bone and cartilage tissues in the treatment of patients with injuries and diseases of the musculoskeletal system. The use of hydrogels for cultivating MSCs will make it possible to protect them after being introduced into the body from to the potentially “hostile” environment of damaged or diseased tissues. To date, the properties of MSCs in their cultivation on matrices based on natural and synthetic materials have not been sufficiently studied.

The aim of this work is to evaluate the toxicity and adhesive properties of hydrogels based on polyvinylpyrrolidone (PVP), obtained by two technologies, for MSCs from human adipose tissue (AT) in vitro.

Material and methods. Four sterile PVP hydrogels were used in the experiment, differing in the technology of preparation, the presence or absence of antibiotics. Toxicity was assessed by the number of vital cells (stained with 0.1% trypan blue solution) after adding the MSC suspension to the gels after 24 h. The behavior of MSCs (AT) was studied in dynamics (on days 2 and 5 of cultivation) in terms of the rate and quality of the formed cell monolayer. Morphological analysis of cells, the state of chromatin in the nucleus, and the presence of cytoplasmic inclusions were performed in samples stained with Giemsa dye.

Results. As a result, it was found that human MSCs (AT) are able to attach to the surface of all 4 matrices, represented by a physicochemically modified artificial material based on PVP. Non-toxicity of all 4 hydrogels and their suitability for short-term cell culture were revealed. MSCs (AT) on PVP hydrogels with the addition of antibiotics showed slower growth, while morphological changes were observed in the form of vacuoles in the protoplasm and pycnosis in the chromatin of the cell nucleus.

Conclusion. Thus, the results obtained can be used for further research and improvement of methods for analyzing the cytotoxicity of the drugs under development.

Currently, for the pharmaceutical development of both original and generic drugs, a systematic scientifically based approach is used to create model formulations and dosage form technology. The use of this approach is expressed in the paradigm of Quality-by-Design (QbD) and the application of quality risk management (QRM) methods. The use of these concepts and their deep integration with each other is due to the presence of uncertainty properties in the manifestation of risks that have different degrees of severity of consequences, the probability of occurrence and their detectability. In the case of the use of original pharmaceutical substances, innovative methods or dosage forms, uncertainty increases, and with them the need to use methods for assessing, analyzing, controlling, accepting and communicating risks. The purpose of this article is to analyze the possibilities for introducing the QRM system into the pharmaceutical development process, identify the project field and critical indicators of quality and process, analyze the features and systematize the initial risk assessment methods, in particular risk identification. The article demonstrates possible methods for assessing and managing risks in pharmaceutical development using solid dosage forms as an example, in particular, in terms of such key characteristics as polymorphism, particle size, solubility of pharmaceutical substances, their interaction with excipients, etc. In addition, when considering risk identification methods such as: brainstorming, Delphi method, interviews, documentation review, application of checklists and construction of cause-and-effect diagrams, etc. an assessment of the advantages and disadvantages of the approaches and the main problems characteristic of all QRM methods was carried out.

Relevance. Authentication of crushed medicinal plant raw materials (RM) is an actual issue because there are species similar in morphology or there is a possibility of interspecific hybridization. The characteristics of the absorption spectra of extracts and their chromatographic profile are significant indicators of the authenticity of RS.

The aim of the study was a comparative analysis by TLC and spectrophotometry of water-alcohol extracts from the herb Thymus marshallianus Willd. (TM) and Thymus serpyllum L. (TP).

Material and methods. Water-alcohol extracts were obtained from 1.0 g of RS (exact suspension) in two ways (extractant – 70% ethanol, heating in a boiling water bath; extractant – 50% ethanol, 20–22 °C). Absorption spectra were recorded in the range of 200–500 nm. Eluent – ethyl acetate : formic acid anhydrous : water (70:15:15) was used for chromatography. Detection of adsorption zones was carried out after treatment with a 5% alcohol solution of AlCl₃ at 365 nm. Rutin, quercetin and cinaroside were used as standard samples.

Results. Microscopic analysis revealed diagnostic signs of the TM and TP leaf – trichomes of various types. The absorption spectra of water-alcohol extracts from TM and TP (obtained with 50% and 70% ethanol) have two pronounced maxima. When AlCl₃ is added, a bathochromic shift of the long-wavelength absorption bands is observed. The chromatograms of these extracts revealed a yellow zone at the level of cinaroside, and differences in the number and color of the adsorption zones of TM and TP extracts.

Conclusion. The absorption spectra of water-alcohol extracts have two pronounced maxima for TP of the same intensity, for TM – different. The intensity of differential absorption spectra of water-alcohol extracts of TM is higher, which indicates a higher content of flavonoids in its extracts. Cinaroside was detected in TM and TP extracts by TLC, while 11 adsorption zones are distinguishable on the chromatogram of extracts from TM herb, and 9 from TP.

Relrvance. Sequoia sempervirens (D.Don) Endl. are the tallest relict long–lived plants characterized by a limited range of growth. Valuable sequoia wood is able to accumulate unique secondary metabolites that do not have synthetic analogues. Sequoia plants grow in specialized microecological niches that limit their natural distribution area. It is known that biotic stress is one of the main environmental factors limiting the introduction of valuable plant species – possible sources of production of unique metabolites. Limiting environmental factors have a direct impact on the growth, development and productivity of secondary plant metabolism. Stress-resistant and highly productive plants can be created using cell biotechnology methods, in particular, in vitro cell selection, which is carried out on a callus culture. Therefore, at the first stage, it is necessary to develop in vitro technology for the rapid production of well-proliferating callus tissue.

Purpose of the study. To study the effect of the mineral composition of the nutrient medium, photoperiod and endogenous polyphenols on the formation of the callus tissue of sequoia (Sequoia sempervirens (D.Don) Endl.) in vitro.

Material and methods. The object of the study was Sequoia sempervirens (D.Don) Endl. plants. Callus tissue was obtained from segments of leaf blades and stem internodes, which were isolated from intact plants. Explants were cultured on MS and WPM nutrient medium containing BAP 2.5 mg/l and 2.5 mg/L 2,4-D. The localization of phenolic compounds was studied in the leaves, stems, apical buds of sequoia plants, as well as in callus tissue obtained on a nutrient medium with different mineral composition and under different lighting conditions. To do this, histochemical methods were used: for the sum of phenolic compounds, the material was stained with 0.08% Fast Blue reagent raster, a reaction with vanillin reagent in hydrochloric acid vapor was used to study the localization of flavans (catechins and proanthocyanidins).

Results. It was found that the applied lighting mode had a significant effect on the intensity of callus tissue formation, its consistency and color. A well-proliferating callus tissue of light yellow color was obtained on two types of nutrient media with no light source. At a 16–hour photoperiod, a dark brown callus tissue was formed, which died during cultivation. As a rule, callus tissue was formed in those places where the localization of phenolic compounds was insignificant. It was found that in the initiated callus cultures grown on the studied nutrient media with no illumination, the content of cells with phenolic compounds was less than that of callus obtained on the same nutrient media, but with the presence of illumination.

Conclusion. Sequoia plants have the ability to synthesize polyphenols, including the flavan series, which is preserved under in vitro conditions. The mineral composition of the studied nutrient media does not significantly affect the biosynthetic ability to form polyphenols in vitro cultures and their growth characteristics.