The study of halophytes as sources of phenolic compounds, as well as conditions that further enhance the accumulation of biologically active compounds in them, is of particular interest. In this paper, the effect of different salinity levels (25–500 mM in the form of NaCl) on the content of phenolic compounds and the antioxidant activity of two rare halophyte species Spergularia marina (L.) Griseb. and Glaux maritima L. cultured in vitro was investigated. A species-specific reaction of plants to salinization was established. In G. maritima, the maximum total content of phenolic compounds was observed at 50–100 mM, flavonoids 75–400 mM, and hydroxycinnamic acids 200–300 mM, as well as individual phenolics (protocatechuic acid, catechin, astragalin, hyperoside, rutin, isoquercitrin, and apigenin derivative) at 100–300 mM NaCl. For S. marina, on the contrary, there was a slight decrease in the content of phenolic compounds when NaCl was added to the nutrient medium compared to the control. The content of protocatechuic acid, rosmarinic acid, and apigenin derivative significantly decreased with increased salt stress. The change in antioxidant activity at different salinity levels was also species specific. The maximum values of different groups of phenolic compounds in G. maritima were observed at 50–300 mM NaCl. The cultivation of S. marina without the addition of NaCl and at 500 mM NaCl allowed the production of plants with the highest content of phenolic compounds. The obtained results can be further used in the development of protocols for the cultivation of these plants in vitro in order to induce the biosynthesis of phenolic compounds in them.

Halophyte plants are known for their resistance to harsh environmental conditions associated with excess salts in their habitats. Their resistance to salinization is due, among other things, to their high ability to detoxify free radicals, owing to the relatively high content of antioxidants. On the coast of the Baltic Sea and in the lagoons, there are several rare halophyte species included in the Red Book of the Kaliningrad Region (Russia) and the Baltic region, such as Spergularia marina (L.) Griseb. and Glaux maritima L. The aim of the research was to study the accumulation of certain groups of phenolic compounds in different parts of S. marina and G. maritima plants under conditions of weak and strong soil salinity, as well as to analyze the antioxidant, antibacterial, and fungicidal activity of extracts of the studied plant species. The present study showed an increase in total phenolic content in the roots and shoots of S. marina, and the shoots of G. maritima, in response to increased soil salinity. At the same time, the total content of flavonoids in all the studied parts of the two plant species remained unchanged. However, the content of individual flavonoids (hesperetin, epicatechin, apigenin derivative, luteolin derivative) in S. marina increased, for G. maritima there was a tendency to reduce the content of flavonoids in roots and shoots with an increase in soil salinity. There was an increase in the total content of hydroxycinnamic acids in the roots of Glaux maritima, as well as an increase in the content of protocatechuic acid in the roots and shoots of Spergularia marina. A positive relationship was established between the antioxidant activity of S. marina root extracts and the total content of phenolic compounds, as well as G. maritima shoots extracts and the total content of phenolic compounds. Extracts of S. marina showed no antibacterial activity against Escherichia coli and Bacillus subtilis, and weak fungicidal activity of stem extracts and inflorescences grown on soils, with high levels of salinities, was detected against Candida albicans. The extracts of roots and shoots from G. maritima showed weak antimicrobial and fungicidal activity.

Callus cultures of medicinal plants have good prospects for biotechnology as sources of bioactive compounds. Hyssop callus cultures (Hyssopus officinalis L.) possess unique chemical composition and properties. To study the factors that enhance the synthesis of bioactive compounds, the author assessed the effect of salinity on the growth, phenolic content, and antioxidant potential in H. officinalis callus cultures. While the content of phenolic compounds and hydroxycinnamic acids was determined using standard methods, the radical absorption activity, reducing capacity, and chelating activity made it possible to measure the antioxidant activity of the callus cultures. High concentrations (200–500 mM) of NaCl suppressed the callus culture growth. The growth agents included three variants: MS-2 (2 mg/L kinetin, 3 mg/L 1-naphthaleneacetic acid), MS-5 (0.8 mg/L 6-benzylaminopurine, 1.5 mg/L 3-indoleacetic acid), and MS-6 (0.2 mg/L 6-benzylaminopurine and 1 mg/L 2,4-dichlorophenoxyacetic acid). The nutrient medium salinity affected the yield of phenolic compounds in the MS-2 callus culture. As the nutrient medium salinity increased, the amount of phenolic compounds went down. Adding 50 and 100 mM NaCl to MS-5 and MS-6 nutrient media boosted the content of phenolic compounds in the callus culture. The total content of hydroxycinnamic acids pointed at a significant decrease in their amount at different NaCl concentrations. Various salt concentrations in the nutrient medium inhibited the antioxidant activity of the callus culture extracts. In this research, salt stress failed to increase the accumulation of phenolic compounds and hydroxycinnamic acids in H. officinalis. callus culture extracts. It also proved ineffective as antioxidant activity catalyst.

Hyssopus officinalis L. is a valuable medicinal plant. Its extracts have good commercial prospects as biologically active substances. Biotechnological production methods that yield valuable secondary metabolites often involve callus cultures, which differ in qualitative and quantitative content of target substances. This research compared phenolic secondary metabolites, antioxidants, and antimicrobial activities in extracts of three callus cultures in vitro, native plants, micro-plants and a commercial preparation of H. officinalis. The extracts were obtained using 70% ethanol. The content of phenolic compounds, flavonoids, hydroxycinnamic acids, and tannins was determined by standard methods. The method of high-performance liquid chromatography revealed the content of individual phenolic compounds. The antioxidant activity tests relied on DPPH, FRAP, and ABTS. The antimicrobial and fungicidal activity was assessed by the disc-diffusion method. The Murashige-Skoog medium with 0.2 mg/L 6-benzylaminopurine and 1 mg/L 2,4-dichlorophenoxyacetic acid demonstrated a high growth index of callus crude biomass (11.6 ± 1.4). The same culture was rich in phenolic compounds (35.5 ± 1.2 mg-eq. gallic acid/g solids), hydroxycinnamic acids (82.5 ± 2.6 mg-eq. rosmarinic acid/g solids), tannins (49.6 ± 0.8 mg-eq. gallic acid/g solids), rosmarinic acid (20.25 ± 1.84 mg/g solids), and caffeic acid (1.48 ± 0.08 mg/g solids). Its extracts showed significant antioxidant activity according to DPPH (127.8 ± 5.6 mg-eq. ascorbic acid/g solids) and FRAP (15.9 ± 1.3 mg-eq. ascorbic acid/g solids). However, they had a rather weak antibacterial and fungicidal activity against Bacillus subtilis (7.2 ± 0.2 mm) and Candida albicans (7.0 ± 0.2 mm) at a concentration of 2 mg/disk. The antibacterial and fungicidal activity against Escherichia coli was equally weak at 1 mg/disk (7.0 ± 0.3 mm) and 2 mg/disk (7.3 ± 0.1 mm), respectively. The extracts of H. officinalis callus cultures and micro-plants in vitro produced the best yield of secondary metabolites compared to the native plant extract. Biotechnological methods proved effective in obtaining valuable compounds from cell cultures.

In recent decades, interest in halophyte plants has increased due to their high content of biologically active substances with powerful antioxidant, antimicrobial, anti-inflammatory and antitumor properties and promising for the prevention of various diseases. Several species of halophytes grow on the territory of the Kaliningrad region, among which the rare species Spergularia marina (L.) Griseb. is of particular interest, the biological activity and content of secondary metabolites of which have not been studied sufficiently. The purpose of this study was to obtain callus cultures, to study the content of some groups of phenolic compounds and the antioxidant activity of extracts. The study carried out the selection of growth regulators and concentrations that induce callus formation. 19 nutrient media were selected for the induction of S. marina callus cultures. The conducted phytochemical analysis showed a significant content of phenolic compounds and hydroxycinnamic acids, as well as a high level of antioxidant activity of extracts of callus cultures. Out of 19 callus cultures, cultures obtained on Murashige and Skoog nutrient media containing the following combinations of growth regulators are promising for obtaining target secondary metabolites: 0.25 mg/l TDZ and 1 mg/l 2,4-D; 0.1 mg/l TDZ and 1.5 mg/l 2,4-D; 0.25 mg/l TDZ and 0.25 mg/l IBA; 0.5 mg/l TDZ and 0.25 mg/l IBA; 0.25 mg/l KinN and 0.5 mg/l 2,4-D.

Hyssopus officinalis L. is a valuable essential oil plant of the Lamiaceae family, which owes its pharmacological properties to phenolic compounds, flavonoids, and hydroxycinnamic acids. Cultivation in vitro makes it possible to control morphogenesis and the accumulation of secondary metabolites by adding growth regulators to the nutrient medium. Since this requires knowledge of optimal concentrations, the aim of this in vitro study was to investigate the effect of cytokinin-type growth regulators on the micropropagation and content of bioactive substances in H. officinalis.The research featured in vitro microplants of H. officinalis (cv. Lekar) . The seeds were sterilized using H ₂ O ₂ , AgNO ₃ , NaClO, and H ₂ SO ₄ as disinfectants. They were then sown on a Murashige and Skoog medium without growth regulators. The resulting seedlings were cultured on an medium supplemented with different concentrations of cytokinins (6-benzylaminopurine, kinetin, thidiazuron, and 6-(γ,γ-dimethylallylamino)purine). The experiment lasted for 30 days under a 16/8-h light/dark regimen at 25 ± 1°C. The obtained microplant extracts underwent spectrophotometry to determine the total contents of phenolic compounds, flavonoids, hydroxycinnamic acids, and antioxidant activity. Statistical data processing was performed using a one-way analysis of variance and hierarchical cluster analysis.The treatment of H. officinalis seeds with concentrated sulfuric acid resulted in 98% sterility and 99% germination, exceeding previously published data. Among the growth regulators, 6-benzylaminopurine at 0.1 mg/L was found to be the most effective for morphogenesis, resulting in the highest shoot length (4.6 cm), shoot number (3.2), and node number (9.6). Cluster analysis confirmed that concentrations of 0.1 mg/L 6-benzylaminopurine, 1.0 mg/L kinetin, and 0.1 mg/L thidiazuron were optimal for microplant development. In terms of bioactivity, 6-benzylaminopurine (0.1 mg/L) maximally increased the content of phenolic compounds (5-fold), hydroxycinnamic acids (5-fold), and antioxidant activity, while thidiazuron (0.1 mg/L) selectively stimulated flavonoid accumulation.The novel methods of seed sterilization combined with specific growth regulators for H. officinalis ensured the production of plant material with a targeted biochemical profile.