Chemical Investigation and Antinociceptive Activity Evaluation of Marrubium Vulgare L. Aqueous Extract.

Marrubium vulgare L. (Lamiaceae) has a long history of use in traditional herbal medicine for the treatment of respiratory tract infections, inflammatory conditions, and pain. This study aimed to investigate the chemical composition, acute toxicity, and antinociceptive effects of the aqueous extract from M. vulgare leaves (AEMV). Antioxidant activity was evaluated using DPPH and reducing power assays. The chemical composition of AEMV was determined through LC-MS/MS, and the levels of total phenolics, flavonoids, and condensed tannins were quantified. Acute oral toxicity was assessed in male Swiss mice with a single oral dose of AEMV (1, 2, 5 g/kg). The analgesic impact was examined through writhing, hot plate, and formalin tests. Our findings not only confirmed the safety of the extract in animal models but also revealed significant antioxidant activity in AEMV. High-performance liquid chromatography (HPLC) analysis identified important bioactive compounds, with marrubiin being a major component. Furthermore, AEMV demonstrated robust antinociceptive properties in all conducted tests, highlighting its potential as a valuable natural source of bioactive compounds suitable for a wide range of therapeutic applications.

Marrubium vulgare Extract Improves Spatial Working Memory and Oxidative Stress Damage in Scopolamine-Treated Rats.

Background: The cholinergic neuronal loss in the basal forebrain and increasing brain oxidative stress are one of the main features of the brain suffering from Alzheimer's disease. Marrubium vulgare (M. vulgare), commonly known as 'white horehound,' possesses a variety of valuable properties, such as antioxidative, anti-inflammatory, and antidiabetic activities. Moreover, it possesses neuromodulatory properties that could potentially impact short-term memory functions. Objective: The present study was undertaken to investigate the preventive effects of water M. vulgare extract on working memory, cholinergic neurotransmission, and oxidative stress in rats with scopolamine (Sco)-induced dementia. Methods: Male Wistar rats (200-250 g) were divided into four experimental groups. The plant extract was administered orally for 21 days, and Sco (2 mg/kg) was administered intraperitoneally for 11 consecutive days. The behavioral performance of the animals was evaluated by the T-maze test. The effect of the extract on acetylcholinesterase (AChE) activity and antioxidant status in cortex and hippocampus were also monitored. Results: Our experimental data revealed that treatment with M. vulgare significantly increased the percentage of correct choices of rats with Sco-induced dementia in the T maze test (by 38%, p < 0.05). Additionally, it reduced AChE activity in the hippocampus (by 20%, p < 0.05) and alleviated oxidative stress induced by Sco, particularly in the cortex. Conclusions: M. vulgare water extract demonstrated working memory preserving effect in rats with Sco-induced dementia, AChE inhibitory activity and in vivo antioxidant potential, and deserve further attention.

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.

Marrubium persicum Improved the Biological Parameters Associated with Angiogenesis and Inflammation in Mice.

The genus Marrubium, belonging to the family Lamiaceae, is highly praised in herbal medicine of different countries for having renowned healing properties. Herein, the anti-inflammatory and anti-angiogenesis potential of Marrubium persicum methanol extract was evaluated in a mouse air pouch model of inflammation. Aerial parts of M. persicum were solvent extracted using the Soxhlet apparatus. Subsequently, air injections were performed (for 3 days) into the mice's backs to bring about an air pouch, while carrageenan was used to induce inflammation. The mice were divided into four groups, including; negative control (normal saline into the pouch), control (carrageenan), treatment and positive control (dexamethasone). The inflammatory markers were analyzed 48h after injecting carrageenan, and a haemoglobin assay kit assessed the quantification of angiogenesis in granulation tissue. M. persicum methanol extract at doses of 3.5, 5, 7.5 and 10 mg/kg represented significant decreases in inflammatory parameters. Compared to the control group, the optimum dose (3.5 mg/kg) lessened the myeloperoxidase (MPO) and angiogenesis activity, as well as haemoglobin levels. In sum, the methanol extract of M. persicum exhibited anti-inflammatory effects against carrageenan-induced inflammation, which could be related to its antioxidant and inhibitory effects on neutrophils' infiltration.

Chemical Profile and Cytotoxicity Evaluation of Aerial Parts of Marrubium vulgare L. From Different Locations in Turkey.

Marrubium vulgare L. (Lamiaceae) is used for respiratory and gastrointestinal system disorders in folk medicine. According to European Pharmacopoeia criteria, standardization of the plant is defined by its marrubiin content. In present study, phenolics, marrubiin and essential oil compositions of M. vulgare from different locations in Turkey were analyzed quantitatively by UPLC, GC and GC/MS. Besides, their cytotoxic potentials were evaluated. In the samples, forsythoside B (77-400 mg/100 g dw), arenarioside (forsythoside F) (0-241 mg/100 g dw), verbascoside (acteoside) (171-416 mg/100 g dw) and apigenin-7-O-glucoside (0-17 mg/100 g dw) were determined in different ranges. Marrubiin contents (0.58-1.46 %) of some samples were two times higher than European Pharmacopoeia standards (0.7 %). ß-Caryophyllene (7.24-20.34 %), (Z)-ß-farnesene (1.58-34.85 %), germacrene D (9.8-13.37 %), bicyclogermacrene (1.71-8.63 %) and ß-bisabolene (0-16.68 %) were detected as major compounds in essential oils. The sample from the west of Aegean Region showed cytotoxicity against human neuroblastoma (SH-SY5Y) cell lines (IC50 : 59.80 µg/mL) although it has no effect on non-cancerous NIH-3T3cell lines. This is the first report on phenolic profiles of M. vulgare populations from Turkey. Their potential as marrubiin source for pharmaceutical industry should be considered.

Antioxidant, Antimicrobial and In Silico NADPH Oxidase Inhibition of Chemically-Analyzed Essential Oils Derived from Ballota deserti (Noë) Jury.

Ballota deserti (Noë) Jury (B. deserti) is a medicinal plant used in Ayurvedic medicine. The chemical composition, antioxidant, antibacterial, and antifungal properties of essential oils from B. deserti (EOBD) against drug-resistant microorganisms were examined in this work. Hydrodistillation was used to extract EOBD, and gas chromatography-mass spectrometry was used to identify its constituents. Ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and total antioxidant capacity (TAC) were used to assess the antioxidant effect of EOBD. The disc diffusion agar and the microdilution tests were used in the assessment of the antibacterial properties of EOBD against clinically resistant pathogenic microorganisms. An in silico approach was used to evaluate the inhibitory potential of EOBD against NADPH oxidase. The yield of EOBD was 0.41%, and was primarily composed of linalool (37.82%), cineole (12.04%), and borneol (11.07%). EOBD had good antioxidant potency, with calculated values of 19.82 ± 1.14 µg/mL (DPPH), 64.78 ± 5.21 µg/mL (FRAP), 996.84 ± 20.18 µg EAA/ mg (TAC). Both Gram-negative and Gram-positive bacteria were inhibited by EOBD with inhibition zones ranging from 17.481.75 mm to 28.471.44 mm. EOBD exhibited MICs ranging from 10.78 g/mL to 22.48 g/mL when tested against bacteria using the minimum inhibitory concentration (MIC) assay. Similarly, impressive antifungal activity was observed against fungal strains with inhibition zone widths ranging from 16.761.83 to 36.791.35 mm. Results of MICs assay against fungi showed that EOBD had MICs values ranging from 15.32 ± 1.47 to 23.74 ± 1.54 µg/mL. Docking results showed that thujone, o-cymene, and butanoic acid contained in EOBD possessed strong activity against NADPH oxidase, with glide scores of -5.403, -5.344, and -4.973 Kcal/mol, respectively. In light of these findings, the EOBD may be seen as a potential source of chemical compounds with significant biological capabilities that can be advantageous as natural antioxidants and develop an effective weapon against a wide range of pathogenic bacteria.

Chemical Composition and In Vitro Antioxidant and Antimicrobial Activities of Marrubium vulgare L.

In this study, the polyphenol content and the antioxidant and antimicrobial activities of hydroethanolic (MVE) and hydroacetonic (MVA) leaf extracts of Marrubium vulgare L. were examined. The results indicated that the total phenolic content was higher in MVA (112.09 ± 4.77 mg GAE/DW) compared to MVE extract (98.77 ± 1.68 mg GAE/DW). The total flavonoid content was also higher in MVA extract (21.08 ± 0.38 mg QE/g DW) compared to MVE (17.65 ± 0.73 mg QE/g DW). Analysis of the chemical composition revealed the presence of 13 compounds with a total of 96.14%, with the major compound being malic acid (22.57%). Both extracts possess a good total antioxidant activity. DPPH and FRAP assays indicated that the MVE extract possesses a better antioxidant activity, with IC50 = 52.04 µg/mL ± 0.2 and EC50 of 4.51 ± 0.5 mg/mL, compared to MVA extract (IC50 = 60.57 ± 0.6 µg/mL and EC50 of 6.43 ± 0.0411 mg/mL). Moreover, both extracts exhibited strong antimicrobial activity against certain nosocomial strains as indicted by the MIC values, which ranged between 0.93 mg/mL and 10 mg/mL. Taken together, these results reveal the importance of M. vulgare as a natural antioxidant with important antimicrobial activity.

HPLC-Analysis, Biological Activities and Characterization of Action Mode of Saudi Marrubium vulgare against Foodborne Diseases Bacteria.

The present study aims to evaluate the chemical composition, metabolites secondary and pharmacology activities of methanolic extract of Marrubium vulgare collected from King Saudi Arabia. Moreover, the primary mode of action of the tested extract was studied here for the first time against E. coli and L. monocytogenes. HPLC analysis shows that the major components in the tested extract are luteolin-7-O-d-glucoside, ferulic acid and premarrubiin. Obtained data demonstrated that the investigated extract was richer in phenol (26.8 ± 0.01 mg/GAE g) than in flavonoids (0.61 ± 0.05 mg EC/mL). In addition, the methanolic extract showed an important antioxidant capacity against the DPPH (IC50 = 35 ± 0.01 µg/mL) and ABTS (IC50 = 25 ± 0.2 µg/mL) radical scavenging and a strong inhibition of acetylcholinesterase enzyme with an IC50 value corresponding to 0.4 mg/mL. The antibacterial activity demonstrated that the evaluated extract had significant activity against both Gram-positive and Gram-negative bacteria. The effect of time on cell integrity on E. coli and L. monocytogenes determined by time-kill and bacteriolysis tests showed that the M. vulgare extract reduced the viability of both strains after 8 and 10 h and had a bacteriolytic effect against two different categories of bacteria, Gram-positive and negative, which are not of the same potency. Based on obtained data, it can be concluded that Saudi M. vulgare has a high pharmacological importance and can be used in preparation of food or drugs.

Chemical diversity analysis and biological activities evaluation of different polyphenolic extracts of Marrubium deserti De Noé from Algeria.

ETHNOPHARMACOLOGICAL RELEVANCE: Marrubium deserti De Noé (Lamiaceae) commonly known as desert horehound, is a small perennial herb distributed in arid climates. This plant has been used in Algeria for the treatment of cold, cough, fever and inflammation. AIM OF THE STUDY: This study aimed to perform a phytochemical analysis and to evaluate the effect of diethyl ether (ExDiEth), n-butanol (ExBut) and hydroalcoholic (ExHyD) extracts of the aerial parts of M. deserti De Noé on inflammation, nociceptive response and fever in animal models. MATERIALS AND METHODS: The total phenolic content and the different phenolic fraction (flavones-flavonols, free aglycones, anthocyanidins, C-glycosides and flavonic heterosides) were quantified in order to study the chemical diversity. The anti-inflammatory effect was evaluated using carrageenan-induced paw oedema test and the nociceptive response was determined by the chemical method using acetic acid-induced writhing response and formalin-induced paw licking tests and by the thermal method using the hot plate in mice. In addition, anti-fever was investigated using yeast-induced pyrexia in rats. RESULTS: Quantitative analysis revealed that among all extracts, ExBut had a higher phenolic content and the aerial parts of M. deserti De Noé collected from two different sites (Biskra and Tamanresset) had a significant difference in the contents of flavonic heterosids and flavones-flavonols. PCA and CA showed a strong intra-specific variability of M. deserti De Noé individuals. Our results showed that the ExDiEth, ExBut and ExHyD treatments prevented the increase of paw oedema by 56.25%, 66.65% and 45.56% inhibition percent, respectively. Moreover, treatments with these extracts significantly (p<0.05) decreased the number of writhing and increased the reaction time to thermal stimulation as well as demonstrated reduction in paw licking time both at early and late phases. Likewise, they also showed a significant reduction of yeast-induced pyrexia till 4 h of treatment. CONCLUSION: Results from this study show that M. deserti De Noé aerial parts exhibited anti-inflammatory, analgesic with central and peripheral actions and antipyretic activities which could be associated with the presence of some phytochemicals as flavones, flavonols, flavonic heterosids and phenolic acids.

In Vitro and In Vivo Studies of Metabolic Activation of Marrubiin, a Bioactive Constituent from Marrubium Vulgare.

Marrubiin, a furanoid compound, is a well-known diterpenoid lactone isolated from Marrubium vulgare, which displays a wide spectrum of pharmacological effects and potential hepatotoxicity. Considering that marrubiin contains a structural alert, furan ring, metabolic activation may be one of the major metabolic pathways, and the reactive metabolite may be involved in the hepatotoxicity. The present study was carried out to investigate the bioactivation mechanism of marrubiin in rats and humans. Marrubiin was initially metabolized into cis-butene-1,4-dial intermediate, which was readily trapped by glutathione (GSH) and N-acetyl-lysine (NAL) in the microsomal incubations supplemented with NADPH. A total of nine conjugates were detected and identified by high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. M1-M3 and M6 and M7 were characterized as mono-GSH conjugates, and M4 and M5 were identified as bis-GSH conjugates. M8 and M9 were identified as NAL conjugates. In rat bile, five GSH conjugates (M1-M3; M6 and M7) were detected. M1, M8, and M9 were chemically synthesized, and their structures were characterized by 13C NMR. Sulfaphenazole, ticlopidine, and ketoconazole displayed significant inhibitory effect on the bioactivation of marrubiin. Further phenotyping revealed that CYP2C9, CYP2C19, and CYP3A4 were the primary enzymes catalyzing the bioactivation of marrubiin. The current study provides evidence for the CYP-dominated bioactivation of marrubiin to the corresponding cis-butene-1,4-dial intermediate, which enables us to better understand the potential side effects caused by marrubiin.

Phytochemical Analysis and Trypanocidal Activity of Marrubium incanum Desr.

The rationale inspiring the discovery of lead compounds for the treatment of human parasitic protozoan diseases from natural sources is the well-established use of medicinal plants in various systems of traditional medicine. On this basis, we decided to select an overlooked medicinal plant growing in central Italy, Marrubium incanum Desr. (Lamiaceae), which has been used as a traditional remedy against protozoan diseases, and to investigate its potential against Human African trypanosomiasis (HAT). For this purpose, we assayed three extracts of different polarities obtained from the aerial parts of M. incanum-namely, water (MarrInc-H2O), ethanol (MarrInc-EtOH) and dichloromethane (MarrInc-CH2Cl2)-against Trypanosoma brucei (TC221), with the aim to discover lead compounds for the development of antitrypanosomal drugs. Their selectivity index (SI) was determined on mammalian cells (BALB/3T3 mouse fibroblasts) as a counter-screen for toxicity. The preliminary screening selected the MarrInc-CH2Cl2 extract as the most promising candidate against HAT, showing an IC50 value of 28 µg/mL. On this basis, column chromatography coupled with the NMR spectroscopy of a MarrInc-CH2Cl2 extract led to the isolation and identification of five compounds i.e. 1-α-linolenoyl-2-palmitoyl-3-stearoyl-sn- glycerol (1), 1-linoleoyl-2-palmitoyl-3-stearoyl-sn-glycerol (2), stigmasterol (3), palmitic acid (4), and salvigenin (5). Notably, compounds 3 and 5 were tested on T. brucei, with the latter being five-fold more active than the MarrInc-CH2Cl2 extract (IC50 = 5.41 ± 0.85 and 28 ± 1.4 µg/mL, respectively). Furthermore, the SI for salvigenin was >18.5, showing a preferential effect on target cells compared with the dichloromethane extract (>3.6). Conversely, stigmasterol was found to be inactive. To complete the work, also the more polar MarrInc-EtOH extract was analyzed, giving evidence for the presence of 2″-O-allopyranosyl-cosmosiin (6), verbascoside (7), and samioside (8). Our findings shed light on the phytochemistry of this overlooked species and its antiprotozoal potential, providing evidence for the promising role of flavonoids such as salvigenin for the treatment of protozoal diseases.

Marrubium vulgare L.: A Phytochemical and Pharmacological Overview.

Marrubium vulgare is a plant with high bioactive potential. It contains marrubiin, a labdane diterpene that is characteristic for this genus, as well as a complex mixture of phenolic compounds. According to numerous studies, M. vulgare acts as a good antioxidant agent, and due to this, it could potentially be useful in treatments of cancer, diabetes mellitus, and liver diseases. In addition, its anti-inflammatory, wound-healing, antihypertensive, hypolipidemic, and sedative potential are discussed. Apart from that, its antimicrobial activity, especially against Gram+ bacteria, fungi, herpes simplex virus, and parasites such as Toxoplasma gondii, Trichomonas vaginalis, and Plasmodium berghei-berghei was recorded. Additionally, it could be used as a chicken lice repellent, herbicide, and natural insecticide against mosquito larvae and natural molluscicide. In veterinary medicine, M. vulgare can be used as an anthelmintic against the eggs and larvae of bovine strongyles parasites, and as an antibiotic against bovine mastitis caused by resistant bacterial strains. Due to the mentioned benefits, there is a tendency for the cultivation of M. vulgare in order to ensure high-quality raw material, but more firm scientific evidence and well-designed clinical trials are necessary for the well-established use of M. vulgare herb and its preparations.

Antioxidant and antifungal activities of marrubiin, extracts and essential oil from Marrubium vulgare L. against pathogenic dermatophyte strains.

OBJECTIVE: Medicinal plants extracts and plant-derived compounds are one of the natural sources for discovering new antifungal agents, the objectives of this work were to investigate for the first time the antidermatophytic, antipathogenic activities of methanol, acetone extracts, and essential oil of Marrubium vulgare L. grown in Tunisia and its active compound marrubiin on pathogenic for animals and humans, such as some dermatophytes and pathogenic for plants, and to evaluate antioxidant activities of different extracts with consideration to their chemical compositions. MATERIAL AND METHODS: Acetone and methanol extracts were evaluated by HPLC, the essential oil was also analyzed by GC/MS. PCL assay was used to determine the antioxidant activity. RESULTS: Results showed that methanol and acetone extracts exhibited a significant antioxidant activity (261.41 and 272.90µmol TE/g respectively), while the lowest one was observed in the case of marrubiin and essential oil. The antifungal activity of different extracts, marrubiin and essential oil at two concentrations (20 and 100µg/mL) were screened against the dermatophytes fungi Microsporum gypseum, Microsporum canis, Arthroderma cajetani, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum and against two fungi strains (Botrytis cinerea, Pythium ultimum). Among tested extracts, marrubiin at 100µg/mL showed about 50% inhibition for T. mentagrophytes and E. floccosum. The anti-phytopathogenic activity was also carried out, only marrubiin had in activity against B. cinerea at the highest dose (32.40%), while methanol extract of M.vulgare and marrubiin are able to increase the mycelial growth of P. ultimum at the highest concentration (45.15 and 40.30% respectively). CONCLUSION: In our study, we conclude that M.vulgare and marrubiin can be used as natural antioxidants and antifungal agent for treatment of skin dermatophyte infections.

Cytotoxic activity of methanolic fractions of different Marrubium spp. against melanoma cells is independent of antioxidant activity and total phenolic content.

The Marrubium genus (horehound) has proved to be an abundant source of biologically active compounds, but there is little knowledge about its potential anticancer activity. Moreover, some Marrubium species have not been the subject of study in this regard. In this study, we performed comparative analysis of phenolic acid (PhA) content and total phenolic content in fractions obtained from methanolic extracts of Marrubium vulgare L. (common horehound), Marrubium cylleneum Boiss. & Heldr. and Marrubium friwaldskyanum Boiss herbs. We examined the cytotoxicity of these fractions against a human melanoma cancer cell line (A375) and normal human skin fibroblasts (BJ) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test, cell cycle analysis and real-time monitoring of cell viability. We detected caffeic, p-coumaric, ferulic and gentisic acids among the PhAs. Although the extracts obtained demonstrated low total phenolic content and did not show significant antioxidative properties, the nonhydrolyzed PhA fraction exhibited cytotoxic activity against a human melanoma cancer cell line, without affecting normal fibroblasts. Both acidic and alkaline hydrolysis abolished this activity, indicating that the esterified forms of phenolic compounds caused the observed cytotoxic effects. Further investigation of these compounds may facilitate the development of novel drugs for cancer treatment.

Functional characterization of the cytochrome P450 monooxygenase CYP71AU87 indicates a role in marrubiin biosynthesis in the medicinal plant Marrubium vulgare.

BACKGROUND: Horehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds. RESULTS: Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare. CONCLUSIONS: In a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.

The use of a microwave-assisted solvent extraction coupled with HPLC-UV/PAD to assess the quality of Marrubium vulgare L. (white horehound) herbal raw material.

INTRODUCTION: Marrubium vulgare is a herbal remedy presents in several European Pharmacopoeias and commonly marketed as white horehound. The chemotaxonomic marker of Marrubium genus is marrubiin and its content may change in response to biotic and abiotic stress. OBJECTIVE: Development of a microwave-assisted solvent extraction (MASE) methodology suitable for exhaustively extracting marrubiin from M. vulgare leaves, easily applicable to large sets of samples. Evaluation of the influence of copper(II) on marrubiin production. MATERIAL AND METHODS: M. vulgare leaves were dried, extracted exploiting MASE and analysed via high-performance liquid chromatography ultraviolet photodiode array detection (HPLC-UV/PAD) system. A design of experiments approach was adopted to select the best extraction conditions. Extraction parameters (solvent composition, extraction time and temperature), were studied applying two full factorial experimental designs in a sequential approach. To analyse samples, a rapid HPLC-UV/PAD method was set up. RESULTS: The best results in terms of marrubiin extraction yield were obtained extracting samples at 120°C with 100% ethanol, for 15 min (3 × 5 min microwave cycles). The developed methodology was successfully applied to matrices grown in Greenhouse conditions and under stress induced by copper(II), selected as model agent for abiotic stress. Progressively decreasing production of marrubiin was evidenced in connection with treatment with 80, 200 and 300 mg/L copper sulphate. CONCLUSION: An efficient methodology for the extraction and determination of the amount of marrubiin in large sets of samples of M. vulgare plants was developed. Results demonstrated that marrubiin is an easily detectable marker useful for evaluating M. vulgare reaction to stress.

Evaluation of antioxidant capacity of endemic plant Marrubium astracanicum subsp. macrodon: Identification of its phenolic contents by using HPLC-MS/MS.

Antioxidant properties of Marrubium astracanicum subsp. macrodon solvent extracts were measured by both cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) methods. According to the results, ethanol extract of the plant has high potential of reducing antioxidant activity on CUPRAC method. However, water extract of the plant has lower antioxidant potential. Furthermore, both water and ethanol extracts showed lower reducing antioxidant activity compare to standards on FRAP method. Moreover, the composition and content of plant leaves were detected by UHPLC-ESI-MS/MS. High concentrations of quinic acid, p-coumaric acid and malic acid were determined.

Determinación estructural de flavonas presentes en el extracto metanólico de hojas de Marrubium vulgare L. "Cordón" / Structural determination of flavones presents in the methanolic extract of leaves of Marrubium vulgare L.. "Cordón"

Objetivo. Determinar la estructura química de flavonas aisladas del extracto metanólico de hojas de Marrubium vulgare L. "Cordón", mediante comparación con lo publicado por TJ Mabry. Materiales y métodos. Se elaboró extracto metanólico de hojas de Marrubium vulgare L. "Cordón" . Se determinó su solubilidad en solventes de polaridad creciente. Se detectaron los componentes químicos utilizando tricloruro férrico, reactivo de Shinoda, gelatina, entre otros reactivos cromogénicos. Se realizó cromatografía en capa fina y por espectroscopía UV/VIS se propusieron estructuras químicas para los metabolitos tipo flavonas presentes en el extracto metanólico de hojas de Marrubium vulgare L. "Cordón" . Resultados. El extracto metanólico de hojas de Marrubium vulgare L. "Cordón" fue soluble en solventes de mediana polaridad. Los metabolitos secundarios encontrados fueron flavonoides, taninos y alcaloides. Se propusieron estructuras químicas de flavonas a través del análisis de los espectros UV/Vis, y por comparación con tablas publicadas en la literatura. Conclusiones. Se determinaron nueve estructuras químicas de metabolitos secundarios tipo flavonas del extracto metanólico de hojas de Marrubium vulgare L. "Cordón" mediante comparación con lo publicado por TJ Mabry.

Objective. Determine the chemical structure of flavones isolated from the methanolic extract of Marrubium vulgare L. "Cordón" leaves by comparison with that published by TJ Mabry. Materials and methods. Methanolic leaf extract of Marrubium vulgare L. "Cordón" was prepared. Its solubility in solvents of increasing polarity was determined. The chemical components were detected using ferric trichloride, Shinoda reagent, gelatin, among other chromogenic reagents. Thin layer chromatography was performed and by UV / VIS spectroscopy chemical structures were proposed for flavone metabolites present in the methanolic leaf extract of Marrubium vulgare L. "Cordón". Results. The methanolic extract of Marrubium vulgare L. "Cordón" leaves was soluble in medium polarity solvents. The secondary metabolites found were flavonoids, tannins and alkaloids. Chemical structures of flavones were proposed through the analysis of the UV / Vis spectra, and by comparison with tables published in the literature. Conclusion. Nine chemical structures of flavone secondary metabolites of the methanolic leaf extract of Marrubium vulgare L. "Cordón" were determined by comparison with that published by TJ Mabry.

In-vitro Anthelmintic Effects of Aqueous and Ethanolic Extracts of Marrubium vulgare Leaves Against Bovine Digestive Strongyles.

OBJECTIVE: The aim of the present study was to evaluate in vitro the anthelmintic activity of Marrubium vulgare L. growing in Algeria against digestive strongyles in naturally infected bovine. METHODS: The anthelmintic activities of the extracts were evaluated using the egg hatch assay and larval mortality assay. Leaves powder of M. vulgare as extracted by maceration. Ethanolic (EE) and aqueous extracts (AE) were tested at 0.78, 1.55, 3.1, 6.2, 12.5, 25, and 50 mg/ml. Albendazole and dimethyl sulfoxide were used as positive and negative controls at concentrations20 mg/ml and 3%, respectively. RESULTS: The mean embryonation rate was maximum in AE and EE (48.4±3.47% and 54.2±2.87%, respectively) of M. vulgare leaves. The extracts of M. vulgare leaves high effects were observed with 50 mg/ml, but the lowest reduction on parasite eggs hatchability was observed in cultures exposed to 0.78 mg/ml to both extracts. The larval mortality rate of both AE and EE from M. vulgare showed that the extracts at 50 mg/ml exhibited 45.8±1.99% and 51±2.53%, respectively, at 24h. CONCLUSION: The findings of the present study showed that AE and EE of M. vulgare leaves have a potential anthelmintic activity on eggs and larvae of bovine strongyles parasites in vitro.

Herbal Supplement Sales in US Increase 8.5% in 2017, Topping $8 Billion. Strongest sales growth in more than 15 years bolstered by continued popularity of Ayurvedic herbs and new formulations of botanicals with general health and nutrition benefits

In 2017, retail sales of herbal dietary supplements in the United States surpassed $8 billion for the first time, reaching an estimated total of $8.085 billion. Consumer spending increased by approximately $633 million, or 8.5%, from 2016 ­ the strongest US sales growth for herbal supplements in more than 15 years. Total US retail sales have increased every year since 2004, and since then, consumer spending on herbal supplements has nearly doubled.