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.

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.

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.

Design of an Epitope-Based Peptide Vaccine Against the Major Allergen Amb a 11 Using Immunoinformatic Approaches.

Allergic diseases are a socially significant problem of global importance. The number of people suffering from pollen allergies has increased dramatically in recent decades. Pollen allergies affect up to 30% of the world population. Pollen of the common ragweed (Ambrosia artemisiifolia L.) is one of the most aggressive allergens in the world. We have used a series of immunoinformatics approaches to design an effective epitope-based vaccine, which might induce a competent immunity against a major allergen Amb a 11. CD8+ and CD4+ T-cell epitopes and their corresponding MHC restricted alleles were identified by prediction tools provided by immune epitope database (IEDB). Among T-cell epitopes, MHC class I peptide (GLMEPAFTYV) and MHC class II peptide (LVCFSFSLVLILGLV) were identified as most suitable. From all predicted B-cell epitopes, only one epitope (GKLVKFSEQQLVDC) containing sequence from the conserved region was chosen for next processing. Selected epitopes have been validated by molecular docking analysis. These epitopes showed a very strong binding affinity to MHC I molecule and MHC II molecule with binding energy scores - 729.3 and - 725.0 kcal/mole respectively. Performed experimental validation showed that only the MHC class II peptide (LVCFSFSLVLILGLV) can stimulate T cells from ragweed allergic patients and IgE antibodies specific to the ragweed pollen do not recognize this epitope. Therefore, this peptide could be potentially used as a vaccine against the major allergen Amb a 11. The B-cell epitope GKLVKFSEQQLVDC forms a stable complex with the IgE molecule (energy weighted score - 695,0 kcal/mole). Tested sera from patients with ragweed allergy showed that the ragweed specific IgE antibodies can bind to the identified B-cell epitope. Population coverage analysis was performed for CD8+ and CD4+ T-cell epitopes. It was predicted that CD4+ T-cell epitope (LVCFSFSLVLILGLV) covers 90.56% of the population of Europe and 99.36% of the world population. CD8+ T-cell epitope (GLMEPAFTYV) has a population coverage of 77.37% for Europe and 71.35% for all the world.

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.