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.

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.

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.

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.

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.

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.

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.

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.

Marrubium vulgare L. Leave Extract: Phytochemical Composition, Antioxidant and Wound Healing Properties.

Several factors contribute in wound generation, e.g., accidental traumas or surgery, and in certain cases, this dermal injury may have a devastating outcome. When skin damage occurs, the human body puts in place a sophisticated choreography, which involves numerous repairing processes to restore physiological conditions. Nevertheless, natural healing mechanisms are ineffective towards chronic or non-healing wounds and thus, therapeutic strategies may represent the only beneficial alternative to counteract these tissue insults. Over the years, numerous studies showed the great potential of plants in promoting wound healing, by virtue of their high contents in antioxidant species. These compounds trigger a molecular cascade that collimate into the promotion of reparative processes. In this article, we report on the potential effect on wound healing of Marrubium vulgare L., a medicinal plant well known for several pharmaceutical activities. To this aim, the methanolic extract was prepared and subjected to a phytochemical investigation, quantifying the amount of marrubiin via NMR and drawing the phytochemical fingerprint via high performance liquid chromatography-ultra violet/photodiode-array detection-electrospray/mass (HPLC-UV/PAD-ESI/MS) analysis. Lastly, the antioxidant properties and wound healing potential have been evaluated.

An investigation on cardioprotective potential of Marrubium vulgare aqueous fraction against ischaemia-reperfusion injury in isolated rat heart.

BACKGROUND: The aim of this study was to evaluate the cardioprotective effects of aqueous fraction of Marrubium vulgare hydroalcoholic extract on cardiac parameters in ischaemic-reperfused isolated rat hearts. MATERIALS AND METHODS: The aerial parts of the plant were extracted with methanol 70% by maceration. The water-soluble portion of the total hydroalcoholic extract was prepared with liquid-liquid extraction (LLE). Afterwards, the antioxidant activity, total phenolic and flavonoids content of the aqueous fraction were determined. In order to evaluate the effects of the aqueous fraction on cardiac parameters and ischaemia-reperfusion (I/R) injury, the Langendroff method was used on male Wistar rats. Harvested hearts were cannulated immediately to the Langendroff apparatus and subjected into 30 min regional ischaemia and 2 h reperfusion, either by a modified Krebs-Henseleit buffer (KHB) solution or enriched KHB solution with plant extract (10, 20, 40 µg/mL). RESULTS: The aqueous fraction was found to be a scavenger of DPPH radical with RC50 value of 47 µg/mL. The total phenolic and flavonoids content of the fraction was 6.05 g gallic acid equivalent and 36.13 mg quercetin equivalent per 100 g of dry plant material. In addition, 40 µg/mL of Marrubium vulgare aqueous fraction significantly decreased infarct size in comparison to control group. All doses considerably reduced the total ventricular ectopic beats during 30 min of ischaemia. The extract at dose of 40 µg/mL noticeably decreased the arrhythmias during the first 30 min of reperfusion. CONCLUSIONS: The results of the study indicated aqueous fraction of Marrubium vulgare possesses a protective effect against I/R injuries in isolated rat hearts.

Intensification of marrubiin concentration by optimization of microwave-assisted (low CO2 yielding) extraction process for Marrubium vulgare using central composite design and antioxidant evaluation.

CONTEXT: Marrubium vulgare Linn (Lamiaceae) was generally extracted by conventional methods with low yield of marrubiin; these processes were not considered environment friendly. OBJECTIVE: This study extracts the whole plant of M. vulgare by microwave assisted extraction (MAE) and optimizes the effect of various extraction parameters on the marrubiin yield by using Central Composite Design (CCD). MATERIALS AND METHODS: The selected medicinal plant was extracted using ethanol: water (1:1) as solvent by MAE. The plant material was also extracted using a Soxhlet and the various extracts were analyzed by HPTLC to quantify the marrubiin concentration. RESULTS: The optimized conditions for the microwave-assisted extraction of selected medicinal plant was microwave power of 539 W, irradiation time of 373 s and solvent to drug ratio, 32 mL per g of the drug. The marrubiin concentration in MAE almost doubled relative to the traditional method (0.69 ± 0.08 to 1.35 ± 0.04%). The IC50 for DPPH was reduced to 66.28 ± 0.6 µg/mL as compared to conventional extract (84.14 ± 0.7 µg/mL). The scanning electron micrographs of the treated and untreated drug samples further support the results. DISCUSSION AND CONCLUSION: The CCD can be successfully applied to optimize the extraction parameters (MAE) for M. vulgare. Moreover, in terms of environmental impact, the MAE technique could be assumed as a 'Green approach' because the MAE approach for extraction of plant released only 92.3 g of CO2 as compared to 3207.6 g CO2 using the Soxhlet method of extraction.

Substitution of Two Active-Site Residues Alters C9-Hydroxylation in a Class†II Diterpene Synthase.

Diterpenes form a vast and diverse class of natural products of both ecological and economic importance. Class II diterpene synthase (diTPS) enzymes control the committed biosynthetic reactions underlying diterpene chemical diversity. Homology modelling with site-directed mutagenesis identified two active-site residues in the horehound (Marrubium vulgare) class II diTPS peregrinol diphosphate synthase (MvCPS1); residue substitutions abolished the unique MvCPS1-catalysed water-capture reaction at C9 and redirected enzyme activity toward formation of an alternative product, halima-5(10),13-dienyl diphosphate. These findings contributed new insight into the steric interactions that govern diTPS-catalysed regiospecific oxygenation reactions and highlight the feasibility of diTPS engineering to provide a broader spectrum of bioactive diterpene natural products.

Marrubium vulgare ethanolic extract induces proliferation block, apoptosis, and cytoprotective autophagy in cancer cells in vitro.

Marrubium vulgare is a European medicinal plant with numerous beneficial effects on human health. The aim of the study was to isolate the plant ethanolic extract (MVE) and to investigate its anti-melanoma and anti-glioma effects. MVE was prepared by the modified pharmacopoeial percolation method and characterized by UHPLC-LTQ OrbiTrap MS. MVE dose-dependently reduced viability of melanoma (B16) and glioma (U251) cells, but not peripheral blood mononuclear cells. It arrested cell cycle in S+G2/M phase, which was associated with the activation of MAP kinase p38 and up-regulation of antiproliferative genes p53, p21 and p27. MVE induced oxidative stress, while antioxidants abrogated its antitumor effect. Furthermore, MVE induced mitochondrial depolarization, activation of caspase-9 and -3, Parp cleavage, phosphatidylserine exposure and DNA fragmentation. The mitochondrial apoptotic pathway was associated with the up-regulation of proapoptotic genes Pten, Bak1, Apaf1, and Puma and down-regulation of antiapoptotic genes survivin and Xiap. MVE also stimulated the expression of autophagy-related genes Atg5, Atg7, Atg12, Beclin-1, Gabarab and Sqstm1, as well as LC3-I conversion to the autophagosome associated LC3-II, while autophagy inhibitors exacerbated its cytotoxicity. Finally, the most abundant phenolic components of MVE, ferulic, p-hydroxybenzoic, caffeic and chlorogenic acids, did not exert a profound effect on viability of tumor cells, suggesting that other components individually or in concert are the mediators of the extracts' cytotoxicity. By demonstrating the ability of MVE to inhibit proliferation, induce apoptosis and cytoprotective autophagy, our results suggest that MVE, alone or combined with autophagy inhibitors, could be a good candidate for anti-melanoma and anti-glioma therapy.

Hepatoprotective activity of white horehound (Marrubium vulgare) extract against cyclophosphamide toxicity in male rats.

The hepatoprotective activity of Marrubium vulgare against cyclophosphamide toxicity in Wistar rats was evaluated. Adult male rats were divided into 4 groups of 6 each: a control group, a group injected with cyclophosphamide (150 mg·kg(-1)) for 3 days, a group orally given a M. vulgare aqueous extract ((500 mg of dry leaves)·kg(-1)·day(-1)) for 30 days then treated with cyclophosphamide, and a group receiving only M. vulgare for 30 days. After 33 days of treatment, activities of alanine amino transferase (ALAT), aspartate amino transferase (ASAT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were determined in serum. Moreover, lipid peroxidation level and superoxide dismutase (SOD) activities, catalase (CAT) and glutathione peroxidase (GPx) were measured in liver. Alterations of these hepatic biomarkers and increased lipid peroxidation confirmed cyclophosphamide-induced liver toxicity. Cyclophosphamide also decreased the enzymatic defense system against oxidative stress. However, when this drug was administered in rats given M. vulgare extract, all the biological parameters underwent much less alteration. Administration of M. vulgare extract was found to be beneficial by attenuating cyclophosphamide-induced liver damage. The protective effect of the plant is mainly attributed to its antioxidant properties and the existence of phenolic acids and flavonoids, as highlighted by HPLC-based analysis.

An Insight into a Blockbuster Phytomedicine; Marrubium vulgare L. Herb. More of a Myth than a Reality?

Aerial parts and the root of Marrubium vulgare L. (white horehound) have been traditionally used in Europe and in southern and eastern Mediterranean countries. During colonization, the plant was introduced in America to great levels of popularity because of the simplicity of its growing; it was especially popular in Mexico and Brazil, where it has been known as 'maromba', 'marroio' or 'marroio-branco'. Ethnopharmacological uses of M. vulgare include treating respiratory diseases such as acute or chronic bronchitis, colds and asthma. The plant is also used in cases where there is a lack of appetite or dyspepsia and for diagnosed type II diabetes. It has even been used for antihypertensive therapy. For decades, scientists have carried out extensive research trying to explain these and other pharmacologic actions. It is time to systematize and critically analyse the quality of results found to date. Copyright © 2016 John Wiley & Sons, Ltd.

Antioxidant activity and total phenolic and flavonoids content variations of leaves extracts of white Horehound (Marrubium vulgare Linné) from three geographical origins.

OBJECTIFS: To elucidate the effect of the sampling location of Marrubium vulgare L. leaves on phenolic contents and antioxidant proprieties of flavonoids extracts. MATERIALS AND METHODS: M. vulgare L. leaves were collected from three different geographical locations belonging to northwest Algeria: Tessala (mountain region), M'sila forest (coastal region), and Ain Skhouna (steppe region). The flavonoid extraction was achieved using organic solvents with different polarities (methanol, chloroform, ethyl acetate, and hexane). Folin-Ciocalteu colorimetric method was used for quantification of total phenolic contents, and aluminum chloride assay for quantification of total flavonoid contents. The antioxidant properties of flavonoids extract were studied by free l,l-diphenyl-2-picrylhydrazyl radical-scavenging technique. RESULTS: Total phenolic and flavonoids concentrations varied respectively between 40.7 and 160mg gallic acid equivalents/g and 27.4 and 66.3mg catechin equivalents/g. The DPPH free radical-scavenging activity shows that the antioxidant activity of the flavonoid extracts varied significantly (P<0.001) depending on the type of the organic solvent used, and the sampling location. The methanol, chloroform and ethyl acetate extracts exhibited the highest percentages of inhibition unlike to the aqueous and hexane extracts. These percentages are ranged from 54.8 to 98.8% at 1000µg/mL. In general, M'sila forest flavonoids extracts showed the highest free radical inhibition capacity; followed by those of Ain Skhouna and Tessala Mountain. The inhibitory concentration 50 (IC50) ranged from 33.7 to 774µg/mL and often exceeded those recorded by phenolic standards (ascorbic acid, gallic acid, caffeic acid, tannic acid and catechin). The phytochemical screening revealed the presence of some flavonoid classes, such as flavans and flavanols. CONCLUSION: The results suggested a potent antioxidant activity of M. vulgare flavonoids extracts, which may find its application in feature research for the food and the pharmacological industries.

Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare.

Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual ßα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.