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.

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.

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.

Recently, compounds of plant origin have been the focus of increased scientific interest. Micromeria frivaldszkyana is a rare species endemic to Bulgaria, whose biological activity remains unknown. This article aims to evaluate the subchronic toxicity of Micromeria frivaldszkyana methanolic extract and its effect on cognition in rats. Following 90 days of oral administration, a histopathological evaluation of brain, kidney, and liver tissues was conducted. Additionally, serum levels of total bilirubin (TB), conjugated bilirubin (CB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), uric acid (UA), and urea (U) were measured. Cognitive function was studied after 7 d of treatment using activity cage test, along with tests for active memory, passive memory, anxiety, spatial and working memory, and explorative activity. The experiments showed no toxic effects of the extract in subchronic application. No adverse effects on brain function were observed after 14 days of treatment. While the extract increased the motor activity of the animals, it did not significantly improve the learning and memory processes. In conclusion, the methanolic extract of Micromeria frivaldszkyana in doses 250 and 500 mg/kg bw did not induce toxicity after 90-day treatment in rats. These doses did not significantly affect central nervous system (CNS) functions, although increased motor activity was observed after 14 days of treatment with the extract.

Betonica bulgarica is an endemic species distributed in Bulgaria. The chemical composition of the essential oil analysed by GC–MS (Gas chromatography–mass spectrometry) and the content of trace elements analysed by ICP–MS (Inductively coupled plasma mass spectrometry) were determined. Additionally, a study on the types and distribution of trichomes was done using a microscope with a camera. The essential oil was characterized using a high concentration of sesquiterpene hydrocarbons, whose major compounds are β-caryophyllene (17.4%), germacrene D (9.9%), and β-bourbonene (6.7%). The contents of manganese (177.2 µg/g) and strontium (156.8 µg/g) were highest among the investigated micronutrients. Two types of trichomes were identified on the adaxial and abaxial epidermises of the leaves of B. bulgarica—covering and glandular. Peltate stacked glandular trichomes with a four-celled head of type B were observed on the leaf surface.

The genus Marrubium (Lamiaceae) is widely used in traditional medicine. While some representatives of the genus have been well investigated, the biological activity of others remains largely unknown. The aim of the current study was to assess the in vitro antioxidant potential and the effect on the reactivity of isolated rat gastric smooth muscles (SM) of aqueous extracts of Marrubium friwaldskyanum inflorescences, stems and leaves, and Marrubium peregrinum as a whole herb. The antioxidant activity was analyzed through multiple spectrophotometric and fluorometric assays. The effect on SM reactivity was determined by the treatment of excised gastric muscles of 10 male Whistar rats with the plant extracts alone or successive to 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide, ketanserin, verapamil, and acetylcholine. According to the obtained results, the M. friwaldskyanum leaf extract exhibited the greatest antioxidant potential, followed by the M. peregrinum one. The SM reactivity analysis revealed that the treatment with all four extracts induced a dose-dependent contractile response with predominant cholinergic character. However, activation of serotoninergic and/or dopaminergic pathways was also observed. Furthermore, when applied after verapamil, the extracts showed a SM relaxant effect. The discovered biological activity will serve as a basis for future analyses through which the therapeutic effect of the plants will be investigated.