Analyzing the Antioxidant Activity and Fatty Acid Composition of Monofloral Mullein (Verbascum sp.) Pollen Oil obtained via Various Extraction Techniques.

This study focused on characterizing fatty acids and evaluating the antioxidant properties in oils extracted from mullein (Verbascum sp.) bee-collected pollen, utilizing soxhlet and ultrasound-assisted methods with acetone and hexane solvents. Soxhlet extraction demonstrated high efficiency in mullein bee pollen oil extraction. The highest levels of total phenolic content (TPC), total flavonoid content (TFC), DPPH⋅, and ABTS⋅+ activities (41.07±1.43 mg GAE/g extract; 1.86±0.01 mg QE/g extract; 16.23±0.68 mg TE/g extract; 56.88±0.43 mg TE/g extract, respectively) were observed in oil extracted using the soxhlet method with acetone solvent. Conversely, ultrasound-assisted extraction with hexane yielded oils rich in saturated fatty acids, while acetone extraction contained higher monounsaturated fatty acids. Palmitic, linoleic, and oleic acids were predominant in the extracted oils. This study introduces, for the first time, the identification of fatty acids found in mullein bee pollen oil, along with an examination of their antioxidant properties. The choice of solvent was found to significantly influence compound extraction compared to the extraction method.

In vitro antimicrobial and antioxidant activities, essential oil composition, and in silico molecular modeling analysis of secondary metabolites from roots of Verbascum sinaiticum.

Verbascum sinaiticum is locally used to treat wound, stomachache, viral infection, cancer, sunstroke fever, abdominal colic, diarrhea, hemorrhage, anthrax, and hepatitis. The objective of this study was to identify the compounds and to evaluate the antimicrobial and antioxidant activity of the extracts and isolated compounds from V. sinaiticum. The 1H-NMR, 13C-NMR, and DEPT-135 were used to elucidate the structures of isolated compounds. Essential oils were extracted by hydrodistillation method and their chemical analyses were performed by GC-MS. The broth microdilution method was used to evaluate the antimicrobial activity. The radical scavenging activity of the extracts and isolated compounds were evaluated using DPPH method. Silica gel column chromatographic separation of root extracts afforded seven known compounds: 3'-(4''-methoxy phenyl)-3'-oxo-propionyl hexadecanoate (1), harpagoside (2), pulverulentoside I (3), scrophuloside B4 (4), scropolioside A (5), scropolioside-D2 (6), and harpagide 6-O-ß-glucoside (7), which are all reported from this species for the first time. The EO extracts from leaves and roots were the most susceptible to Streptococcus agalactiae, with a 2 mg/mL MIC. The EO from roots was effective against Candida albicans and Trichophyton mentagrophytes, with a MIC of 8 mg/mL. The MeOH and CH2Cl2/CH3OH (1:1) root extracts showed the maximum activity against S. epidermidis with MIC values of 0.25 mg/mL. The strongest antibacterial effects were demonstrated against Staphylococcus epidermidis, which exhibited a 0.0625 mg/mL MIC for compound 1. The strongest radical scavenging activity was exhibited by the methanol extract (IC50 = 3.4 µg/mL), and compounds 4, 6, 5, 3, 7, and 2 with IC50 values of 3.2, 3.38, 3.6, 3.8, 4.2, and 4.7 µg/mL, respectively, in comparison with ascorbic acid (IC50 = 1.3 µg/mL). The results of the molecular docking analysis of compounds revealed minimal binding energies range from -38.5 to -43.1 kJ/mol, -33.1 to -42.7 kJ/mol, -34.7 to -39.3.7 kJ/mol, -25.5 to -37.6 kJ/mol against human myeloperoxidase (PDB ID: 1DNU), murA enzyme (PDB ID: 1UAE), human topoisomerase IIß (PDB ID: 4fm9), S. epidermidis FtsZ (PDB number: 4M8I) proteins, respectively. The docking results and the in vitro antibacterial activity are in good agreement. These findings show that the isolated compounds 2-7 can act as potential antioxidants and strong antibacterials against Staphylococcus aureus and S. epidermidis. As a result, V. sinaiticum root extracts have the potential to be effective in treating diseases caused by bacteria and free radicals, as long as further investigation has been suggested for the ultimate decision of this plant's potential candidate.

Ferruginoside†D: A Novel Phenylethanoid from Verbascum leiocarpum.

A novel phenylethanoid, ferruginoside D, together with fourteen known compounds were isolated from the Verbascum leiocarpum for the first time. Chemical structures of isolated compounds were determined by spectroscopic analysis including HR-ESI-MS and NMR spectra. The antioxidant, α-glucosidase inhibitory properties, and antiproliferative activities against multiple cell lines (A172, C6, HeLa, A2780, SW620, HT29, Beas2B, RPE, and HSF) of the isolated compounds were evaluated in vitro. According to the results, iridoids, flavonoids (luteolin and apigenin), and phenylethanoids (poliumoside and isoverbascoside) with strong antiproliferative potential were also found to have cytostatic effects. Furthermore, the investigation revealed that compounds luteolin, poliumoside, verbascoside, isoverbascoside, ferruginoside C, ferruginoside D, and ursolic acid show potent alpha-glucosidase inhibitory activity, while compounds luteolin, verbascoside, and isoverbascoside exhibit substantial antioxidant activity. The new compound (ferruginoside D) was found moderately active against cancer cell lines, with strong alpha-glucosidase inhibitory activity, and moderate antioxidant properties.

Bioactive Compounds of Verbascum sinuatum L.: Health Benefits and Potential as New Ingredients for Industrial Applications.

Verbascum sinuatum (V. sinuatum) is a plant belonging to the Scrophulariaceae family that has been used as an ingredient in traditional medicine infusions for the treatment of many diseases. The aerial part of this plant is a source of bioactive compounds, especially polyphenols and iridoids. Moreover, antioxidant activity studies have shown that V. sinuatum phenolic and flavonoid composition is higher than those in other plants of the same genus. V. sinuatum bioactive compound composition could vary according to the harvesting location, growing conditions of the plants, sample preparation methods, type and concentration of the extraction solvent, and the extraction methods. The obtention of these compounds can be achieved by different extraction techniques, most commonly, maceration, heat assisted extraction, and infusion. Nevertheless, since conventional extraction techniques have several drawbacks such as long times of extraction or use of large amounts of solvents, the use of green extraction techniques is suggested, without affecting the efficiency of the extraction. Moreover, V. sinuatum bioactive compounds have several biological activities, such as antioxidant, anticancer, cardiovascular, antimicrobial, antidiabetic, and neuroprotective activities, that may be increased by encapsulation. Since the bioactive compounds extracted from V. sinuatum present good potential as functional food ingredients and in the development of drugs or cosmetics, this review gives an approach of the possible incorporation of these compounds in the food and pharmacological industries.

Isolation, characterization and anticancer activity of secondary metabolites from Verbascum speciosum.

Herein, two iridoid glucosides aucubin (1) and ajugol (2), and two phenyl ethanoids, verbascoside (3) and poliumoside (4) were isolated from the methanol extract of the aerial parts of Verbascum speciosum and used to study about their anticancer activity for the first time. The structures of all compounds were elucidated using spectroscopic data (IR, 1D and 2D NMR, LC-TOF/MS). Antiproliferative activities of Aucubun (1) and Verbascoside (3) were tested against A-549 (human colon cancer), MDA-MD-453 (human breast cancer) and 3T3-L1 (mouse fibroblast)cell lines by XTT assay. In addition, the anticarcer mechanism of action of aucubin (1) was investigated on MDA-MB-453 cells for the first time. XTT result showed that both applied compounds exhibited antiproliferative effect at different dose ranges depending on the cancer type, as well as selectivity between cancer and healty cell lines. Flow cytometry analyzes revealed that aucubin (1) exerts its cytotoxic effect in MDA-MB-453 cells by directing cells to early apoptosis and inhibiting the P13K/AKT signaling pathway.

Antioxidant, AChE inhibitory, and anticancer effects of Verbascum thapsus extract.

Verbascum thapsus (VT) is a medicinal plant that is used in folk medicine to treat a variety of ailments. For this study, the biological functions of VT methanol extract were determined in vitro. The plant's methanol extract was created through the maceration process. The phytochemical composition of plant extracts was investigated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The antioxidant capacity of the extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazil) and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) tests and its cytotoxicity was assessed using the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)) assay on the Caco-2 (human colorectal adenocarcinoma cells), LNCaP (Lymph Node Carcinoma of the Prostate), and HEK293 cell lines (Human embryonic kidney 293 cells) used to model colon, prostate, and non-cancerous cells. VT extract showed low DPPH and ABTS radical scavenging activities compared to standard antioxidants at 30 mg/ml concentration. In addition, it was determined that VT extract inhibited acetylcholinesterase enzyme.

Health-promoting and disease-mitigating potential of Verbascum thapsus L. (common mullein): A review.

Medicinal plants represent rich sources of traditional medicines and numerous currently used medicines are either directly or indirectly derived from plants. Verbascum thapsus L. (great mullein or common mullein), a medicinal herb indigenous to northern Africa, western and central Asia, and Europe, has been brought to the Americas and Australia. It has been used as a medicine for lung, skin and throat disorders and has a long history of therapeutic importance, particularly as an astringent and calming agent. Presently, the dried leaves, flowers, various plant extracts and flower oil are used in several formulations within Indian traditional medicine. An extract taken from the roots is useful in minimizing toothache, and it also relieves stiffness and seizures. V. thapsus contains a wide variety of phytoconstituents, such as flavonoids, iridoid, phenylethanoid and phenylpropanoid glycosides, saponins, as well as vitamin C and minerals. The most valuable constituents are coumarin and hesperidin, which possess healing properties. Emerging literature based on experimental studies on V. thapsus demonstrates various biological and pharmacological properties, including antiviral, antioxidant, analgesic, sedative, anti-inflammatory, hypnotic, antibacterial, antifungal, as well as anticancer activities. The present review provides an updated, comprehensive, and critical evaluation of various health-promoting and disease-mitigating properties of V. thapsus.

Phenolic and flavonoid compounds and antioxidant activity in flowers of nine endemic Verbascum species from Iran.

BACKGROUND: In this study, flowers of nine mullein species (Verbascum erianthum, V. songaricum, V. speciosum, V. szovitsianum, V. stachydiforme, V. sinuatum, V. haussknechtianum, V. cheirantifolium, and V. saccatum) were collected from Iran. The total flavonoid content (TFC), antioxidant activity, total carotenoid (TCC), ß-carotene content and the amount of phenolic compounds were determined in all of the collected mullein species. Quantitative analysis of phenolic compounds was performed using high-performance liquid chromatography with diode-array detection (HPLC-DAD). RESULTS: Verbascum saccatum and V. songaricum, in particular, were found to be rich in total flavonoids and exhibited the highest antioxidant capacity. The HPLC-DAD analysis revealed that V. erianthum possessed the highest amount of caffeic acid (0.022 g kg-1 DW), chlorogenic acid (2.649 g kg-1 DW), p-coumaric acid (0.253 g kg-1 DW), and apigenin (0.066 g kg-1 DW). The highest gallic acid (0.134 g kg-1 DW), rutin (5.254 g kg-1 DW), quercetin (1.303 g kg-1 DW), and cinnamic acid (1.031 g kg-1 DW) content were observed in V. saccatum. Our results demonstrate the considerable variations in the TFC, antioxidant activity, TCC, and ß-carotene content among the mullein species. CONCLUSIONS: The findings of this study provide useful information for breeding strategies, and for choosing the best species with high phenolic compound content to produce natural antioxidants for medical and pharmaceutical use. © 2021 Society of Chemical Industry.

Evaluation of antioxidant, cytotoxic, antibacterial effects and mineral levels of Verbascum lasianthum Boiss. ex Bentham.

The present study aimed to determine the antibacterial, antioxidant, cytotoxic activities and element levels of Verbascum lasianthum Boiss. ex Bentham. The free radical scavenging activity, total phenolic content, total antioxidant capacity, total oxidant capacity levels were analyzed as the antioxidant parameters. Seven bacteria and one yeast strains were used to determine the antimicrobial activity. The cytotoxic effects of plant extracts were determined using A549, MCF-7, HepG2 and SH-SY5Y cell lines. The findings demonstrated that the antioxidant activity increased with an increase in the phenolic content of extracts. This species is rich in bio-elements such as Fe, Cu, Mn, Zn, and Mg. Different concentrations of extracts could have antibacterial activity. This plant had an apparent cytotoxic effect only in the A549 cell line and increased the proliferation in other cell lines. The findings demonstrated that plant could be used alone or as a supplement to the current treatment protocols in diseases due to their antioxidant, antibacterial and cytotoxic effects. However, it is recommended that Verbascum L. species intended for use in therapy should be procured from areas where there is no soil pollution or organic farming is preferred.

The effect of the Verbascum Thapsus on episiotomy wound healing in nulliparous women: a randomized controlled trial.

BACKGROUND: The pain and discomfort caused by episiotomy affect the quality of life of the mothers, so rapid and complete repair of the episiotomy is very important. Due to the effective ingredients of Verbascum Thapsus, it has been used since ancient times to treat wounds. Therefore, this study aimed to evaluate the effect of Verbascum Thapsus on episiotomy wound healing. METHODS: The study was designed as a randomized, double-blind, controlled clinical trial. Ninety-three primiparous women who were referred to Fatemeh Zahra Hospital in Saveh in 2015 were randomly divided into two groups of intervention (Verbascum Thapsus) and control (placebo). Both groups covered the episiotomy wound twice a day for 10 days with 2 cm of prescribed creams. Wound healing was assessed using the REEDA scale before the intervention and on days 1,3 and 10 after the intervention. RESULTS: Before the intervention, there was no statistically significant difference in terms of demographic characteristics, obstetrics, and REEDA scores between the two groups (p < 0.05). The mean scores of REEDA on days 1 and 3 in the intervention group were better than the control group but were not statistically significant. However, on the tenth day after the intervention, the mean scores of REEDA were significantly better in the Verbascum group than the placebo (p = 0.01). CONCLUSIONS: According to the results of this study, it seems that Verbascum Thapsus is effective in repairing episiotomy wounds. The researchers hope that the results of this study can provide clinical evidence for the use of this herbal medicine in the wound healing process. TRIAL REGISTRATION: This study was registered in the Iranian Registry of Clinical Trials (IRCT) with the code " IRCT201404073106N15 " on 02/12/2015.

Secondary metabolites from Verbascum bugulifolium Lam. and their bioactivities.

Five iridoid glycosides catalpol (1), specioside (2), ajugol (3), ajugoside (4), 8-O-acetylharpagide (5), two phenylethanoid glycosides, verbascoside (6) and glucopyranosyl-(1→Gi-6)-martynoside (7), four flavonoids, luteolin (8), luteolin 7-O-ß-glucopyranoside (9), luteolin 7-O-rutinoside (10), apigenin 7-O-rutinoside (11), quercetin 3-O-rutinoside (12) and ß-sitosterol (13) were isolated from the aerial parts of Verbascum bugulifolium Lam. for the first time. Their structures were elucidated by NMR and MS experiments. The extracts, and the isolates were evaluated for their in vitro antioxidant (DPPH•, ABTS• and CUPRAC), anti-inflammatory (LOX inhibition) and antimicrobial activities. Compounds 6 and 8 showed the highest antioxidant activity in all tests, where luteolin (8) showed the relatively best anti-inflammatory activity compared to other samples with 54.1 ± 5.0% inhibition at 1 µg/mL. All the tested compounds showed weak antimicrobial activity against tested microorganisms. This is the first phytochemical and bioactivity study on V. bugulifolium.[Figure: see text].

Micromorphology, pharmacognosy, and bio-elemental analysis of an important medicinal herb: Verbascum thapsus L.

The present study was conducted on characterization of morpho-anatomical, phytochemical, and bio-elemental analysis of root, stem, and leaf of Verbascum thapsus. Morphologically Verbascum is a biennial plant that flowers for a month and a half in mid- to late summer. Various organoleptic features of root, leaf, and stem were recorded. Anatomically the T. S of the root, stem, and leaf showed a typical dicot histological differentiation. Leaf possessed anomocytic stomata, crescent shape vascular bundles, and covered with long and stellate type trichomes while, stem contained collateral type of vascular bundles and a well-developed pith to store phytochemicals responsible for various pharmacological activities. The powder drug study through scanning electron microscopy revealed the presence of various types of tissues. Branched, tree like and stellate trichomes in root and leaf help in absorption and reduce loss of water. These anatomical features are responsible for the survival of the plant as biennial. Four macro elements (Na, K, Ca, and Mg) and seven microelements (Cr, Cu, Fe, Mn, Ni, Zn, and Cd) and their concentrations in ppm were also studied using Atomic Absorption Spectroscopy. Phytochemical screening of methanolic extract showed existence of various secondary metabolites, while mucilage and anthraquinones was not detected. The present study helps to understand the taxonomic identification of the plant based on morpho-anatomical features and throws the attention of the researchers to carry out the work for developing its various formulations, which can ultimately be beneficial for the human beings as well as animals.

Inhibition of Amyloid ß Aggregation Using Optimized Nano-Encapsulated Formulations of Plant Extracts with High Metal Chelator Activities.

BACKGROUND: The role of Fe+2, Cu+2 and Zn+2 in facilitating aggregation of Amyloid ß (Aß) and consequently, the progression of Alzheimer's disease (AD) is well established. OBJECTIVE: Development of non-toxic metal chelators is an emerging era in the treatment of AD, in which complete success has not been fully achieved. The purpose of this study was to determine plant extracts with high metal chelator and to encapsulate them in nano-micellar systems with the ability to pass through the Blood Brain Barrier (BBB). METHODS: Extracts of 36 different Anatolian plants were prepared, total phenolic and flavonoid contents were determined, and the extracts with high content were examined for their Fe+2, Cu+2 and Zn+2 chelating activities. Apolipoprotein E4 (Apo E) decorated nano-formulations of active extracts were prepared using Poly (Lactide-co-Glycolide) (PLGA) (final product ApoEPLGA) to provide BBB penetrating property. RESULTS: Verbascum flavidum aqueous extract was found as the most active sample, incubation of which, with Aß before and after metal-induced aggregation, resulted in successful inhibition of aggregate formation, while re-solubilization of pre-formed aggregates was not effectively achieved. The same results were obtained using ApoEPLGA. CONCLUSION: An optimized metal chelator nano-formulation with BBB penetrating ability was prepared and presented for further in-vivo studies.

Chemical constituents and HRESI-MS analysis of an Algerian endemic plant - Verbascum atlanticum batt. - extracts and their antioxidant activity.

This is the first report on the phytochemistry and antioxidant activity of ethyl acetate and n- butanol extracts from an Algerian endemic plant Verbascum atlanticum Batt. (Scrophulariaceae). Both extracts were subjected to a phytochemical study by semi-preparative HPLC, which led to the isolation and identification of nine compounds: methyl linolenate (1), methyl linoleate (2), Phytol-1(3), Martynoside (4), Isomartynoside (5), Cis-martynoside (6), Ilwensisaponin C (7), Ilwensisaponin B (8), Ilwensisaponin A (9). In addition, the fractions from both extracts were analysed by LC-UV-MS and HRESI-MS. This later revealed the presence of eight other metabolites by using a comparison with known microbial metabolites data. Finally, both extracts were estimated for their phenolic and flavonoid contents as well as the evaluation of their antioxidant activity using five different assays DPPH, CUPRAC, reducing power, ß-carotene bleaching and superoxide DMSO alkaline. The results showed that the ethyl acetate extract had the most antioxidant effect.

Phenolic compounds and bioactive properties of Verbascum glabratum subsp. bosnense (K. Malý) Murb., an endemic plant species.

Detailed analysis of phenolic composition and antioxidant and antimicrobial activities of Verbascum glabratum subsp. bosnense (K. Malý) Murb., an endemic species of southeastern Dinaric Alps was performed for the first time. The phenolic composition measured via UHPLC-MS/MS of four extract with different polarity suggested this plant species is very rich in both phenolic acids and flavonoids. Ethanol extract was chemically the most versatile containing 12 compounds with quercitrin and rosmarinic acid as the majors, while water extracts were rich in 4-hydroxybenzoic acid, salicylic acid, morin, and apigenin. All extracts showed high antioxidant potential measured spectrophotometrically with IC50 values ranging 0.139 - 0.021 mg/mL. Antimicrobial testing using agar diffusion test showed that ethanol extract was the most potent against all tested organisms. Also, these activities are correlated with the content of phenolic compounds, which suggest they are active ingredients of the extracts.

The Antioxidant, Anticarcinogenic and Antimicrobial Properties of Verbascum thapsus L.

BACKGROUND: The antioxidant, anticarcinogenic, and antimicrobial activities of Verbascum Thapsus L., known as great mullein, (an important medicinal plant containing different biochemical compounds including sesquiterpenes, flavonoids, saponins and lignins) were determined. There is not much data, to our knowledge, in this respect. METHODS: The antioxidant activities of V. Thapsus were investigated by the DPPH (2, 2- diphenyl- 1-picrylhydrazyl) method. Using GC-MS, the presence of different anticarcinogenic products including 1-hexzanol (2.11%), 2-hexene (1.95%), etc. was determined in the ethanolic extract of V. Thapsus. The antimicrobial activities of V. Thapsus were determined by the minimum inhibiting concentration (MIC) and minimum bactericidal concentration (MBC) methods using the Grampositive and -negative bacterial strains. RESULTS: The least concentration of V. Thapsus L. ethanolic extract (50 mg/l) resulted in only 21.26% inhibition of DPPH free radicals, however, the concentrations of 300 mg/l resulted in almost the highest inhibition (91.31%) of DPPH free radicals. The antioxidant activities of synthesized antioxidant BHT at the concentration of 300 mg/l or higher were similar to the antioxidant activities of V. Thapsus L. ethanolic extract. Both the isolated and the standard Gram-negative bacterial strains were more tolerant to the V. Thapsus ethanolic extract, compared with the Grampositive bacterial strains. Bacillus cereus was the most sensitive bacterial strain among the tested bacterial strains. CONCLUSION: The medicinal plant V. Thapsus L. can be used for the treatment of different diseases, such as cancer and infectious diseases.

Biological activities, isolated compounds and HPLC profile of Verbascum nubicum.

Context: Genus Verbascum (Scrophulariaceae) comprises about 360 species of flowering plants. Verbascum has been used in traditional medicine as an astringent, antitussive, analgesic and anti-inflammatory. Objective: Nothing was found in the available literature concerning Verbascum nubicum Murb; therefore, the study evaluates the biological activities, isolated compounds and HPLC profile. Materials and methods: Methanol extract (VME) and butanol fraction (VBF) of air-dried powdered V. nubicum were obtained. Four compounds were isolated from VBE and identified by 1H- and 13C-NMR. High-performance liquid chromatography (HPLC) profile was determined for (VME). LD50, in vitro antioxidant, in vivo antiulcerogenic and anti-inflammatory activities as well as hepatoprotective activity were assessed. Anti-ulcerogenic and hepatoprotective activities were supported by histopathological examinations. Results: HPLC analysis of VME revealed the presence of luteolin 7-glucoside (2215.43 mg/100 g), hesperidin (954.51 mg/100 g) and apigenin (233.15 mg/100 g) as major compounds. Four compounds were isolated and confirmed by NMR data, were identified as gentiopicroside, luteolin, aucubin and gallic acid. The LD50 of VME and VBF extracts were calculated to be 8200 and 4225 mg/kg b.w., respectively. IC50 values of VBE and VMF as measured by DPPH·method were 43.6 and 50 µg/mL, respectively. Also, anti-inflammatory effect of VME (400 mg/kg b.w.) and VBF (200 mg/kg b.w.) induced edema model after 120 min were 61.93 and 56.13%, respectively. Antiulcerogenic activity of VME (400 mg/kg b.w.) and VBF (200 mg/kg b.w.) in albino rats were 65.14 and 84.57%, respectively. Conclusions: The V. nubicum extracts displayed safe and promising antioxidant, anti-inflammatory and hepatoprotective properties. It can be also applied in the pharmacy industry, food industry and healthcare.

Plant-derived verbascoside and isoverbascoside regulate Toll-like receptor 2 and 4-driven neutrophils priming and activation.

BACKGROUND: Neutrophils have a short live in circulation and accelerate greatly local immune responses via increased granulopoiesis and migration at high numbers to infected or inflamed tissue. HYPOTHESIS: Since neutrophils produce a variety of factors with destructive and pro-inflammatory potential the regulation of their homeostasis and functions might be eventually beneficial in inflammation-related pathological conditions. Herein we investigated the effect of natural-derived verbascoside (Verb) and its positional isomer isoverbascoside (IsoVerb) on neutrophil functions. METHODS: We used purified murine bone marrow (BM) neutrophils to study cell responsiveness to priming or activation via Toll-like receptors (TLRs) 2 and 4. The expression of CD11b, chemokine (CXC motif) receptor 2 (CXCR2), the intracellular level of phosphorylated p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF)-α in neutrophils were determined by flow cytometry while the release of macrophage inflammatory protein (MIP)-2 in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that Verb appeared less powerful inhibitor of TLR2 and TLR4-mediated apoptosis than IsoVerb. However at concentrations below 16 µM and in LPS priming conditions Verb was more selective inhibitor of CD11b and CXCR2 expression than IsoVerb. Both compounds showed similar activity on integrin/chemokine receptor expression when neutrophils were stimulated with ZY or were activated with LPS. Verb sustained CXCR2 expression and turnover via regulation of the cell responsiveness to its ligand KC (CXCL1) and via the release of MIP-2 (CXCL2). Both Verb and IsoVerb increased TNF-α production and inhibited p38 phosphorylation in TNF-α+ cells. We fail to discriminate sharply between Verb's and IsoVerb's efficacy when studying p38 phosphorylation in LPS stimulated neutrophils. The multi-parametric analysis provides critical insight on the range of on-target effects of Verb and IsoVerb. CONCLUSION: The strength and selectivity of Verb and IsoVerb depended on the degree of activation and functional state of neutrophils, and both compounds are with potential to affect neutrophil-related pathologies/conditions in heterogenic populations.

High-performance countercurrent chromatographic isolation of acylated iridoid diglycosides from Verbascum ovalifolium Donn ex Sims and evaluation of their inhibitory potential on IL-8 and TNF-α production.

In traditional folk medicine, Verbascum species have been used since ancient times to treat respiratory disorders, hemorrhoids, diarrhea, wounds, eczema and other types of inflammatory skin conditions. Despite the fact that more than 200 bioactive constituents (phenylethanoids, flavonoids, neolignan glycosides, phenolic acids, iridoids, saponins and polysaccharides) have been previously isolated from various Verbascum species, to date preparative high-performance countercurrent chromatography (HPCCC) has never been employed for this purpose. Therefore, in this study, simple HPCCC methods were successfully developed with the aim to primarily isolate acylated iridoid diglycosides from Verbascum ovalifolium Donn ex Sims (oval-leaved mullein). By the use of several biphasic solvent systems containing n-hexane, ethyl acetate, n-butanol/methanol and water, premnacorymboside B (3, 4 mg, 95.4% purity), saccatoside (4, 6 mg, 95.7% purity), premnacorymboside A (7, 6 mg, 98.3%), scorodioside (8, 11 mg, 96.0%) and 6-O-(3'',4''-di-O-trans-cinnamoyl)-α-L-rhamnopyranosylcatalpol (9, 8 mg, 95.3%) were afforded; compounds 7, 8 and 9 have not been previously reported in Verbascum genus. Additionally, two phenolic acids (1, 2), two flavonoids (6, 10) and verbascoside (5) were secondarily isolated. Evaluation of interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) inhibitory properties of the acylated iridoid diglycosides proved that these compounds down-regulated TNF-α release more efficiently than IL-8 secretion. The activity might be dependent on the degree of esterification, as diacyl derivatives showed more potent effects than monoesters. The HPCCC methods herein developed could serve to large scale isolation of constituents from Verbascum genus for extensive biological investigations.

HPLC-DAD-ESI-Q-TOF-MS/MS profiling of Verbascum ovalifolium Donn ex Sims and evaluation of its antioxidant and cytogenotoxic activities.

INTRODUCTION: Plant species of Verbascum genus have been intensively investigated in the last decades but most studies focused on evaluation of their biological activities; there are only few studies dealing with their chemical characterisation. OBJECTIVE: Detailed investigation of the qualitative and quantitative chemical composition, antioxidant and cytogenotoxic activities of a previously non-studied Verbascum species (V. ovalifolium Donn ex Sims). METHODS: Qualitative analysis of secondary metabolites was performed by HPLC-DAD-ESI-Q-TOF-MS/MS, whereas quantitative data were obtained through HPLC-DAD. Antioxidant activity was evaluated using in vitro assays; cytotoxic and genotoxic effects were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) and comet assays, respectively. RESULTS: More than 50 secondary bioactive metabolites belonging to various classes (iridoids, phenylethanoids, flavonoids, phenolic acids) were detected in the methanolic extract of V. ovalifolium and its fractions. The fragmentation pathways of acylated catalpol-type iridoid diglycosides are thoughtfully described herein. The extracts showed good free radical scavenging and ferric ion reducing properties correlated with phenolic, flavonoid, chlorogenic acid and verbascoside contents. Moreover, 24 h treatment of SK-MEL-2 cells with V. ovalifolium extracts produced significant changes in terms of tumour cell viability. The crude extract and the ethyl acetate fraction showed no important signs of cytogenotoxicity in non-tumour cells. CONCLUSION: The performed phytochemical and biological analyses contribute to the preclinical knowledge about V. ovalifolium and they could help exploiting it in novel herbal medicinal products.