Metabolic Composition of Methanolic Extract of the Balkan Endemic Species Micromeria frivaldszkyana (Degen) Velen and Its Anti-Inflammatory Effect on Male Wistar Rats.

Extracts from medicinal plants are widely used in the treatment and prevention of different diseases. Micromeria frivaldszkyana is a Balkan endemic species with reported antioxidant and antimicrobial characteristics; however, its phytochemical composition is not well defined. Here, we examined the metabolome of M. frivaldszkyana by chromatography-mass spectrometry (GC-MS), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS-MS), and inductively coupled plasma mass spectrometry (ICP-MS). Amino acids, organic acids, sugars, and sugar alcohols were the primary metabolites with the highest levels in the plant extract. Detailed analysis of the sugar content identified high levels of sucrose, glucose, mannose, and fructose. Lipids are primary plant metabolites, and the analysis revealed triacylglycerols as the most abundant lipid group. Potassium (K), magnesium (Mg), zinc (Zn), and calcium (Ca) were the elements with the highest content. The results showed linarin, 3-caffeoil-quinic acid, and rosmarinic acid, as well as a number of polyphenols, as the most abundant secondary metabolites. Among the flavonoids and polyphenols with a high presence were eupatorin, kaempferol, and apigenin-compounds widely known for their bioactive properties. Further, the acute toxicity and potential anti-inflammatory activity of the methanolic extract were evaluated in Wistar rats. No toxic effects were registered after a single oral application of the extract in doses of between 200 and 5000 mg/kg bw. A fourteen-day pre-treatment with methanolic extract of M. frivaldszkyana in doses of 250, 400, and 500 mg/kg bw induced anti-inflammatory activity in the 1st, 2nd, and 3rd hours after carrageenan injection in a model of rat paw edema. This effect was also present in the 4th hour only in the group treated with a dose of 500 mg/kg. In conclusion, M. frivaldszkyana extract is particularly rich in linarin, rosmarinic acid, and flavonoids (eupatorin, kaempferol, and apigenin). Its methanolic extract induced no toxicity in male Wistar rats after oral application in doses of up to 5000 mg/kg bw. Additionally, treatment with the methanolic extract for 14 days revealed anti-inflammatory potential in a model of rat paw edema on the 1st, 2nd, and 3rd hours after the carrageenan injection. These results show the anti-inflammatory potential of the plant, which might be considered for further exploration and eventual application as a phytotherapeutic agent.

Chemical Composition and Lipid Bioactive Components of Centaurea thracica Dwelling in Bulgaria.

Centaurea thracica (Janka) Hayek is a plant common in southern Bulgaria. The inflorescences were collected during June and September 2021, while their seeds were obtained in September 2021. The chemical and lipid composition of the inflorescences during the vegetation process of the plant were established. A significant decrease in total proteins (from 8.7 to 7.4%), glyceride oils (2.0-1.7%), and ash (4.5-4.2%) content was observed, while the amount of carbohydrates (72.3-77.2%) and fibers (28.7-35.8%) increased. During the vegetation of the plant, the content of oleic and linoleic acids increased up to 2-3 times, while the level of palmitic acid decreased. The lipids from the seeds were rich in oleic (53.0%) and palmitic (36.2%) acids. The tocopherol content in the oils of the inflorescences during vegetation increased from 58 to 110 mg/kg, and the content in the oil from the seeds was 260 mg/kg. The phospholipid content decreased during vegetation, and differences were observed in the composition between the inflorescences and the seeds. The high content of oleic acid, linoleic acid, tocopherols, and phospholipids determine the nutritional and biological value of the oils isolated from Centaurea thracica, and contribute to their potential use in various directions.

Metabolome Profiling of Marrubium peregrinum L. and Marrubium friwaldskyanum Boiss Reveals Their Potential as Sources of Plant-Based Pharmaceuticals.

Marrubium species have been used since ancient times as food additives and curative treatments. Their phytochemical composition and various pharmacological activities were the focus of a number of scientific investigations but no comprehensive metabolome profiling to identify the numerous primary and secondary metabolites has been performed so far. This study aimed to generate a comprehensive picture of the total metabolite content of two Marrubium species-M. peregrinum and M. friwaldskyanum-to provide detailed information about the main primary and secondary metabolites. In addition, the elemental composition was also evaluated. For this purpose, non-targeted metabolomic analyses were conducted using GC-MS, UPLC-MS/MS and ICP-MS approaches. Nearly 500 compounds and 12 elements were detected and described. The results showed a strong presence of phenolic acids, flavonoids and their glucosides, which are generally of great interest due to their various pharmacological activities. Furthermore, tissue-specific analyses for M. friwaldskyanum stem, leaves and flowers were carried out in order to outline the sources of potentially important bioactive molecules. The results generated from this study depict the Marrubium metabolome and reveal its dual scientific importance-from one side, providing information about the metabolites that is fundamental and vital for the survival of these species, and from the other side, defining the large diversity of secondary substances that are a potential source of phytotherapeutic agents.

Ecological status assessment of Skalenski Lakes (Bulgaria).

Over the past decade new ecological indices based on phytoplankton and macrophytes were developed as part of the tools for assessment of the ecological status of water bodies. This study demonstrates the applicability of two of them (Assemblage index /Q/ and Algae Group Index /AGI/) for evaluation of water bodies from a lake type L4 as well as their comparability. Assessment of the ecological status of two lake ecosystems was performed in order to ensure successful protection, enhancement and management of lowland and semi-mountain lakes in Bulgaria. Data on the aquatic flora from Golyamo Skalensko Lake and Malko Skalensko Lake over a period of two years were used to assess their ecological status. In addition, the toxic potential of the established dominant cyanoprokaryotic species was also evaluated. Phytoplankton- and macrophyte-based metrics resulted in complementary evaluation of temporary and long-term environmental conditions. Despite the hydraulic connection and proximity between the two lakes, Golyamo Skalensko Lake and Malko Skalensko Lake appear as completely different ecosystems, according to the phytoplankton structure (species composition, number of species, abundance, seasonal succession), macrophytes and ecological status.

Current status and prognosis of Raphidiopsis raciborskii distribution in Bulgaria as part of the southeastern region of Europe.

The cyanobacterial species Raphidiopsis raciborskii (Woloszynska) Aguilera et al. has a high invasiveness potential, which in less than a century leads to its cosmopolitan spread. In the temperate climate of Europe, R. raciborskii has been reported in many countries, but there is still a lack of detailed information about the current status of its distribution in lakes of Bulgaria, as a part of the southeastern range of its spread in Europe. We investigated the distribution of the species using data on the phytoplankton of 122 lakes surveyed during 13-years period (2009-2022). The species was found in 33 lakes (up to 324 m asl), and 14 new localities were registered during the studied period. The results reveal that the number of lakes with the presence of R. raciborskii (27 % of all research lakes) and its contribution to the total phytoplankton biomass, has increased significantly over the last decade. The species has successfully adapted and dominated the phytoplankton in 9 lakes, forming a bloom in 8 of them. The dominant position of R. raciborskii causes loss of species and functional diversity of phytoplankton and displaces the native bloom-forming cyanobacteria. Lakes with and without the species were compared based on the available data on bioclimatic and local environmental variables. Statistically significant differences were established with respect to water transparency, conductivity, maximum depth and maximum air temperature in the warmest month. Species distribution models (SDMs) were used to identify lakes in high risk of future invasion by R. raciborskii. The results of the SDMs implementation confirmed the high maximum air temperature and low water transparency to be important predictors of the occurrence of R. raciborskii in freshwater lakes in Bulgaria. In the areas with high summer temperatures the most suitable for R. raciborskii development were found to be shallow polymictic or medium deep lakes with small surface area and low water transparency. In areas with a suitable climate, the large, deep reservoirs with high transparency as well as macrophyte dominated lakes have a low probability of occurrence of R. raciborskii. Future colonization of lakes above 500 m asl (but most likely below 700 m asl) is also possible, especially in the conditions of global warming. SDMs account for climatic and biogeographic differences of lakes and could help in elucidating the underlying factors that control the occurrence and adaptation of R. raciborskii in a given area.

Assessment of the biological activity of Marrubium friwaldskyanum Boiss. (Lamiaceae).

Present scientific evidences about the biological activity and potential medical application of extracts derived from Marrubium friwaldskyanum Boiss. are limited. Therefore, our study was undertaken to define several main characteristics in this regard - in vitro cytotoxicity and antitumor properties, antibacterial activity and immunomodulatory potential. Extracts were obtained from different aerial parts of Marrubium friwaldskyanum - stems, leaves and flowers. The in vitro cytotoxicity and antitumor activity of the samples were evaluated by tetrazolium salt reduction tests and Neutral red uptake assays using four human cell lines (a normal fibroblastic and three adenocarcinoma cell lines/A549, HeLa, HT-29/) and by experiments with HT-29 tumor spheroids. Antibacterial activity toward Gram-negative (Escherichia coli) and Gram-positive (Bacillus cereus) species was assessed based on estimation of minimal inhibitory and minimal bactericidal concentrations as well as longitudinal studies on bacterial viability. Ex vivo assays with normal leukocytes were performed to define potential immunomodulatory activity of the extracts. Our results demonstrated selective antitumor activity of the extracts directed against colon adenocarcinoma HT-29 cells and cervical adenocarcinoma HeLa cell line. Metabolic activity of A549 lung adenocarcinoma cells was affected only by the sample derived from flowers. M. friwaldskyanum leaf and flower extracts showed the highest activity, which included reduction of HT-29 tumor spheroid growth and viability. The studied samples exhibited antibacterial activity against both bacterial species tested. Treatment with M. friwaldskyanum extracts affected specific leukocyte populations (HLA+, CD19+, CD11b+, CD25+ cells). These results demonstrate for the first time complex biological effects of extracts derived from M. friwaldskyanum and their potential to serve as a source of valuable compounds for the pharmaceutical industry.

Antitumor and Immunomodulatory Properties of the Bulgarian Endemic Plant Betonica bulgarica Degen et Neic. (Lamiaceae).

Background: Extracts obtained from different Betonica species have been shown to possess important biological properties. The present study aimed to investigate the cytotoxicity, antitumor and immunomodulatory potential of the endemic plant Betonica bulgarica (Lamiaceae) and thus, reveal new aspects of its biological activity. Methods: Methanolic extract obtained from inflorescences was analyzed for cytotoxicity against mammalian cell lines. The antitumor potential of the sample was determined using human cervical and lung adenocarcinoma cells (HeLa and A549). Programmed cell death-inducing effects against HeLa cells and peripheral blood lymphocytes, as well as immunomodulatory properties of the extract were determined by flow cytometry analysis. Results: The research results demonstrated that the extract has significant inhibitory potential against HeLa cells (mean IC50 value 119.2 µg/mL). The sample selectively induced apoptotic death in tumor cells. Cytotoxic effects towards mouse cell lines were detected following treatment with high concentrations of Betonica bulgarica extract (200 and 250 µg/mL). Twenty-four-hour ex vivo incubation of peripheral blood leucocytes in growth medium containing plant extract induced prominent effects in distinct immune cell populations. They included elevated levels of CD25+ and CD56+ T cells' lymphocytes, particularly CD4+CD25+ and CD8+CD56+ cells. Conclusions: The present study demonstrates that Betonica bulgarica inflorescence extract possesses potential beneficial antitumor and immunomodulatory activity and could serve as a source of bioactive compounds with biomedical application.

Receptor-mediated biological effects of extracts obtained from three Asplenium species.

This study reports the effects of aqueous extracts obtained from three fern species of Bulgarian origin: Asplenium ceterach L., Asplenium scolopendrium L., and Asplenium trichomanes L. on the contractility and bioelectrogenesis of rat gastric smooth muscle tissues. In the concentration range 0.015-0.150 mg/mL the three extracts contracted smooth muscle tissues in a concentration-dependent manner. The contractions caused by A. ceterach L. and A. scolopendrium L. extracts (0.150 mg/mL) were reduced by ketanserin (5 × 10-7 and 5 × 10-6 mol/L), an antagonist of serotonin 5-HT2 receptor. The contraction evoked by A. trichomanes L. (0.150 mg/mL) was significantly reduced by 1 × 10-6 mol/L atropine, an antagonist of muscarinic receptors, and turned into relaxation against the background of 3 × 10-7 mol/L galantamine. After combined pretreatment with galantamine and l-arginine (5 × 10-4 mol/L), this relaxation become more pronounced. The study demonstrates that constituents of A. ceterach L. and A. scolopendrium L. extracts act as agonists of 5-HT2 receptors and cause contraction by activating serotonergic signaling system. A. trichomanes L.-induced reaction is an additive result of two opposite-in-character effects. The dominant contraction is initiated by inhibition of acetylcholinesterase activity. The relaxation develops with pre-inhibited acetylcholinesterase, it is significantly potentiated by l-arginine, and therefore associated with nitrergic signaling pathway.

Selective Anticancer Properties, Proapoptotic and Antibacterial Potential of Three Asplenium Species.

The ferns Asplenium ceterach L., Asplenium scolopendrium L. and Asplenium trichomanes L. have wide application in traditional medicine worldwide. However, the scientific research on their anticancer and antibacterial properties is insufficient. The present article aims to provide more information on this topic. Extracts derived from the aerial parts of A. ceterach, A. scolopendrium and A. trichomanes were examined using a panel of in vitro assays with different bacterial and mammalian cells. The cytotoxicity and anticancer activity of the samples were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays with three human (A549, FL, HeLa) and three murine (3T3, TIB-71, LS48) cell lines. Inhibitory effects on the growth of Gram-positive (Bacillus cereus) and Gram-negative (Pseudomonas aeruginosa) bacteria were determined by the agar diffusion assay. Apoptosis-inducing properties of the extracts were analyzed by flow cytometry. Superoxide dismutase (SOD) activity in extract-treated cells was investigated by ELISA. The obtained results demonstrate selective anticancer activity of all three Asplenium species. The extract from A. ceterach displayed the strongest inhibitory properties against human cervical cancer cells and bacterial cells. It induced a lower level of cytotoxicity against mouse cell lines, indicating a species-specific effect. The extract from A. trichomanes demonstrated better anticancer and antibacterial properties than the sample from A. scolopendrium. Further experiments linked the mechanism of action of A. ceterach extract with oxidative stress-inducing potential and strong proapoptotic potential against the cervical cancer cell line HeLa. A. trichomanes and A. scolopendrium extracts appeared to be potent inducers of necrotic cell death.

Phytochemical Composition, Antioxidant and Antimicrobial Activity of the Balkan Endemic Micromeria frivaldszkyana (Degen) Velen. (Lamiaceae).

The current study investigates the content of sugars, organic acids, phenolic acids and flavonoids, as well as antioxidant and antimicrobial activity of Balkan-endemic Micromeria frivaldszkyana. Glucose was the most abundant sugar in the plant (2.77%), followed by fructose (1.18%) and galactose (0.82%). Eight organic acids were detected with quinic acid being in the highest content-556.3 mg/100 g DW. From the individual phenolic acids, rosmarinic acid was found in the most significant amounts (2040.1 ± 1.97 mg/100 g) and hesperidin was the major representative of flavonoids with content 131.2 ± 5.6 mg/100 g DW. The antioxidant activity of the plant was studied by six methods: 2,2'-diphenylpicrylhydrazyl (DPPH)-286.4 ± 10.43 mM TE/g, 2,2'azinobis (3)-ethylbenzthiazoline-6-sulfonic acid (ABTS)-358.4 ± 10.4 mM TE/g, ferric reducing antioxidant power (FRAP)-388.0 ± 32.4 mM TE/g, cupric reducing antioxidant capacity (CUPRAC)-905.6 ± 19.2 mM TE/g, Oxygen Radical Absorbance Capacity (ORAC)-3250.5 ± 208.1 µmol TE/g and Hydroxyl Radical Averting Capacity (HORAC)-306.1 ± 23.5 µmol GAE/g. In vitro antimicrobial activity against nine microorganism was evaluated but the extract displayed antimicrobial activity only against Listeria monocytogenes ATCC 19111 with inhibition zone diameter 9 mm and minimal inhibitory concentration (MIC) 10 mg/mL.

Naturally present metal ions in plants could interfere with common antioxidant assays.

Most of the commonly applied assays used to assess antioxidant properties of plant extracts exploit the ability of some biologically active metabolites to participate in oxidation-reduction reactions with metal ions. On the other hand, most plants contain different chelated metal ions whose levels depend on the geographic origin, soil, and environmental pollutions. In this study the levels of redox-active metal ions in three plant sources were measured and extracts of these botanicals were treated with ChelexⓇ - an ion exchanger that is noteworthy for its ability to bind transition metal ions. The original and chelated extracts were subjected to three antioxidant assays based on single electron transfer. The results obtained showed statistically significant differences between the original and Chelex-treated extracts suggesting that the naturally present metal ions could interfere with the results of the three most commonly applied antioxidant methods.•The proposed pre-analytical procedure is simple and does not require special instrumental equipment.•Preliminary depletion of redox active metal ions, namely iron and copper ions could improve reproducibility of the analytical methods.•The method allows a more reliable comparison of antioxidant properties of particular botanical species from different geographic regions.

Phytoplankton composition with an emphasis of Cyanobacteria and their toxins as an indicator for the ecological status of Lake Vaya (Bulgaria) - part of the Via Pontica migration route.

As producers of biomass, cyanobacteria are a major part of the phytoplankton in a large number of water basins. Due to the cyanobacterial blooms and cyanotoxins produced, these organisms are recognized as a threat and ecological risk for water bodies. Released cyanotoxins may cause death of many organisms including birds and fish. Vaya Lake is the largest natural lake in Bulgaria. It is located on the Via Pontica migration route of birds between Europe and Africa. Since 2003, the lake has been declared as a "Wetland of international importance" under the Ramsar Convention. According to the literature data from 2002-2006, the Lake is defined as highly eutrophied due to strong anthropogenic pressure, but regular monitoring of the cyanobacterial blooms and presence of cyanotoxins after this period is missing. Taking into account the importance of this unique, protected ecosystem, our aim was to make a complete ecological assessment of the present state of Lake Vaya by using the phytoplankton, with an emphasis on cyanobacterial blooms and the presence of cyanotoxins. As results of the study, we 1) characterized the phytoplankton composition qualitatively and quantitatively; 2) evaluated the ecological status of the western and eastern part of the Lake; 3) identified the potential producers of cyanotoxins; 4) observed cyanobacterial blooms and discussed the influence of macrophytes on their spread; 5) measured the concentrations of the cyanotoxins MCs, CYL and STXs in water samples from both parts of the Lake. Our results indicated the need for continued observation of cyanobacterial composition, blooming and the presence of cyanotoxins in Lake Vaya.

Metabolite Profile and Antioxidant Activity of Some Species of Genus Scutellaria Growing in Bulgaria.

Until now, the interest to plants from genus Scutellaria in Bulgaria has been focused mainly on the terpenes in them. The purpose of this study is to enrich the information on the composition of the Bulgarian Scutellaria species in terms of both polyphenolic content as well as primary metabolites such as mono-, oligosaccharides and organic acids. An aerial part of three Scutellaria species growing in four low mountain regions of Southern Bulgaria was used. The flavonoids scutellarin, baicalin, baicalein, wogonin, wogonoside, luteolin, chrysin and a caffeoyl phenylethanoid glycoside-verbascoside have been identified via HPLC in different extracts from Scutellaria altissima, Scutellaria albida and Scutellaria galericulata. The antioxidant activity of the extracts has been evaluated. The Scutellaria altissima from Mezek and Scutellaria galericulata from Parvenets we studied, which are the richest in flavonoids (represented mainly by baicalin, scutellarin and wogonoside), show the highest Oxygen Radical Absorption Capacity. Hydroxyl Radical Averting Capacity of Scutellaria albida from Mezek and Scutellaria altissima from Bachkovo is the most pronounced, probably due to the content of scutellarin and luteolin and chrysin, respectively. Antioxidant activity of aqueous, methanolic and 70% and 96% ethanol extracts were also determined by the electrochemical method.

Content of phycoerythrin, phycocyanin, alophycocyanin and phycoerythrocyanin in some cyanobacterial strains: Applications.

Cyanobacteria are unique prokaryotes, which are capable to perform oxygenic photosynthesis. Within these organisms, phycobilisomes (PBS) act as an antenna of the photosynthetic pigment apparatus. Phycobilisomes contain several phycobiliproteins (PBP): phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) and phycoerythrocyanin (PEC). The application of phycobiliproteins in the biotechnology, food industry and medicine during the last years is rapidly increasing. The aim of our study was to assess the qualitative and quantitative content of phycoerythrin, phycocyanin, allophycocyanin and phycoerythrocyanin in 14 cyanobacterial strains kept in Plovdiv Algal Culture Collection (PACC) and 4 strains purchased from the Culture Collection of Autotrophic Organisms (CCALA). Our data demonstrated that three strains of Microcoleus autumnalis (PACC 5505, PACC 5522 and PACC 5527) have high potential to produce phycoerythrins (0.132, 0.201 and 0.136 mg/mL, respectively). Similarly, one Microcoleus autumnalis strain (PACC 5522) and one strain of Leptolyngbya boryana (CCALA 084) are suitable for biotechnological production of phycocyanins (0.051 and 0.264 mg/mL, respectively) as well as allophycocyanins (0.102 and 0.171 mg/mL, respectively). In addition, the data about the pigment content could be used as a biochemical marker for taxonomic purposes within the group.

Phylogenetic relationships of some filamentous cyanoprokaryotic species.

The polyphasic approach is the most progressive system that has been suggested for distinguishing and phylogenetically classifying Cyanoprokaryota (Cyanobacteria/Cyanophyta). Several oscillatorialean genera (Lyngbya, Phormidium, Plectonema, and Leptolyngbya) have problematic phylogenetic position and taxonomic state because of their heterogeneity and polyphyletic nature. To accurately resolve the phylogenetic relationship of some filamentous species (Nodosilinea bijugata, Phormidium molle, Phormidium papyraceum), we have performed phylogenetic analyses based on 16S rRNA gene and the phycocyanin operon (PC-IGS) by using maximum-likelihood (ML) tree inference methods. These analyses were combined with morphological re-evaluation. Our phylogenetic analyses support the taxonomic separation of genus Nodosilinea from the polyphyletic genus Leptolyngbya. Investigated Nodosilinea strains always formed a coherent genetic cluster supported with a high bootstrap value. The molecular phylogeny confirmed also the monophyly of the Wilmottia group. In addition, data reveal that although P. papyraceum is morphologically similar to Wilmottia murrayi, this species is genetically distinct. Strains from the newly formed genus Phormidesmis and some Phormidium priestleyi strains were clustered in a separate clade different from the typical Phormidium species, but without strong bootstrap support.