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Sideritis scardica Griseb. is a Balkan endemic species traditionally used for the treatment of pulmonary emphysema and angina pectoris. Recent research has also shown its phytotherapeutic potential as an anticancer and neuroprotective agent. These findings, as well as the endangered status of the species in its wild habitats, have motivated the present research on application of plant cell tissue and organ culture for the purposes of both valuable germplasm conservation and secondary metabolites production. Shoot cultures of the plant were initiated from sterile germinated seeds and the effects of activated charcoal (AC), as well benzyl adenine and 1-naphthaleneacetic acid treatments, were experimented. The phenolic profile analysis was performed by HPLC/DAD/MSn. Comparison with samples collected from wild plants in their natural habitat was performed. It was established that in vitro multiplication induced by plant growth regulators (PGRs) was accompanied by a higher impairment of leaf morphology and trichome formation, as well as by the occurrence of plantlet hyperhydricity and callus formation, as compared with the AC treatments. Shoot culture-derived plant material was shown to produce two phenylethanoids and five flavone glycosides, not detected in the wild collected plant material. In addition, the two types of in vitro culture treatments led to the stimulation of either flavone glycosides or phenylethanoids in the in vitro cultivated plants. Thus, AC stimulated, to a higher extent, flavone glycosides’ accumulation, leading to an elevated flavone/phenylethanoid ratio, as compared with PGR treatments.

Natural deep eutectic solvents (NADES) have been developed in order to replace traditional volatile and toxic organic solvents for the extraction of biologically active substances from natural sources. Recent research has shown that NADES are able to retain and even improve the antioxidant properties of the molecules dissolved in them. The aim of the present study was to evaluate the antioxidant activity of the extracts of two medicinal plants, Plantago major and Sideritis scardica, and of propolis with 10 different NADES. The results obtained confirm that NADES extracts of medicinal plants and propolis have a good antioxidant potential, allowing obtaining natural antioxidants without the application of organic solvents. Because of the biocompatibility and low toxicity of the NADES, the opportunity to apply these extracts directly in formulations, such as cosmetics, food supplements, etc., seems promising.

Nowadays, the environmentally friendly approach to everyday life routines including body supplementation with pharma-, nutraceuticals and dietary supplements gains popularity. This trend is implemented in pharmaceutical as well as cosmetic and antiageing industries by adopting a newly developed green chemistry approach. Following this trend, a new type of solvents has been created, called Natural Deep Eutectic Solvents (NADES), which are produced by plant primary metabolites. These solvents are becoming a much better alternative to the already established organic solvents like ethanol and ionic liquids by being nontoxic, biodegradable, and easy to make. An interesting fact about NADES is that they enhance the biological activities of the extracted biological compounds. Here, we present our results that investigate the potential antiageing effect of CiAPD14 as a NADES solvent and three plant extracts with it. The tested NADES extracts are from propolis and two well-known medicinal plants—Sideritis scardica and Plantago major. Together with the solvent, their antiageing properties have been tested during the chronological lifespan of four Saccharomyces cerevisiae yeast strains—a wild type and three chromatin mutants. The chromatin mutants have been previously proven to exhibit characteristics of premature ageing. Our results demonstrate the potential antiageing activity of these NADES extracts, which was exhibited through their ability to confer the premature ageing phenotypes in the mutant cells by ameliorating their cellular growth and cell cycle, as well as by influencing the activity of some stress-responsive genes. Moreover, we have classified their antiageing activity concerning the strength of the observed bioactivities.

Psoriasis is a chronic inflammatory skin condition characterized by abnormal keratinocyte proliferation and differentiation that is accompanied with dysregulated immune response and abnormal vascularization. Devil’s claw (Harpagophytum procumbens(Burch.) DC. ex Meisn.) tubers extract has been used both systemically and topically for treatment of chronic inflammatory diseases such as arthritis, osteoporosis, inflammatory bowel disease, among others. However, its potential mechanisms of action against psoriasis remains poorly investigated. The human keratinocyte HaCaT cell line is a well-accepted in vitro model system for inflammatory skin disorders such as psoriasis. The present study involved an exploration of the effect of biotechnologically producedH. procumbens(HP) cell suspension extract and pure phenylethanoid glycosides verbascoside (VER) and leucosceptoside A (LEU) in interferon (IFN)-γ/interleukin (IL)-17A/IL-22-stimulated HaCaT cells as a model of psoriasis-like inflammation. Changes in key inflammatory signaling pathways related to psoriasis development were detected by reverse transcription polymerase chain reaction and western blotting. Treatment with LEU, but not VER and HP extract improved psoriasis-related inflammation via suppression of the PI3K/AKT signaling in IFN-γ/IL-17A/IL-22-stimulated HaCaT cells. Our results suggest that LEU may exhibit therapeutic potential against psoriasis by regulating keratinocyte differentiation through inhibition of the PI3K/AKT pathway.

The continent of Antarctica, as a combination of constantly low temperatures, strong winds, short summer season, and high solar radiation, is a highly extreme habitat suggesting appropriate conditions for growth of psychrophilic microorganisms. Five psychrophilic yeast strains were isolated from the samples taken from the region of the Bulgarian Base on Livingston Island, Antarctica: Cryptococcus laurentii AL65, Sporobolomyces salmonicolor AL36, Debaryomyces hansenii, Leucosporidium scotii and Rhodotorula glutinis, and their biomass yield and exopolysaccharides production were investigated. Best growth was observed for L. scotii and C. laurentii AL65, with 7.5 and 6.0 g/L biomass, respectively, and highest exopolysaccharide yield was established for L. scotii. Metabolic profiling revealed phylogenetically based diversity in the identified metabolic profiles. The proton nuclear magnetic resonance (1H NMR) spectroscopy analyses of compounds extracted from the biomass of the strains revealed significant differences in the metabolites between individual yeast strains in our investigation including: among the amino acids alanine, valine, threonine, leucine and tyrosine; some organic acids such as gamma-aminobutyric acid, acetic acid, formic acid and others. Glucose was identified in all investigated strains. The highest diversity of compounds was observed in D. hansenii strain, division Ascomycota. The main compounds in the metabolic profile of Basidiomycota strains were sugars. The statistical analysis revealed significant differences in the studied metabolites among the yeast strains. This result suggests that, together with 16S rRNA gene and enzyme gene analyses, metabolite profiling could be also used as a marker for a phylogenetic distance in fungi evolution.

The pathological manifestation of various diseases can be suppressed by the activation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a transcriptional regulator of the cellular redox balance. Haberlea rhodopensis Friv. is a resurrection plant species endemic for Bulgaria, containing biologically active phenylethanoid glycosides that might possess antioxidant or redox activity. This study aimed to analyze the metabolic profile of in vitro cultured H. rhodopensis and to identify molecules that increase Nrf2 expression in bone marrow neutrophils. Fractions B, D, and E containing myconoside, or myconoside and calceolarioside E in ratios 1:0.6 and 0.25:1 were found to be the most active ones. Fraction B (200 μg/mL) improved neutrophil survival and strongly increased the Nrf2 intracellular level, while D and E, as well as, myconoside and calceolarioside E at the same ratios had a superior effect. Calceolarioside E (32 μg/mL) had stronger activity than myconoside, the effect of which was very similar to that of 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me), used as a positive control. These data indicate that both molecules, used alone or in combination have stimulatory activity on the endogenous Nrf2 level, indicating their therapeutic potential to regulate the cellular redox homeostasis oxidative stress-associated pathologies.

Natural deep eutectic solvents (NADES) are a new alternative to toxic organic solvents. Their constituents are primary metabolites, non-toxic, biocompatible and sustainable. In this study four selected NADES were applied for the extraction of two medicinal plants: Sideritis scardica, and Plantago major as an alternative to water-alcohol mixtures, and the antimicrobial and genotoxic potential of the extracts were studied. The extraction efficiency was evaluated by measuring the extracted total phenolics, and total flavonoids. Best extraction results for total phenolics for the studied plants were obtained with choline chloride-glucose 5:2 plus 30% water; but surprisingly these extracts were inactive against all tested microorganisms. Extracts with citric acid-1,2-propanediol 1:4 and choline chloride-glycerol 1:2 showed good activity against S. pyogenes, E. coli, S. aureus, and C. albicans. Low genotoxicity and cytotoxicity were observed for all four NADES and the extracts with antimicrobial activity. Our results confirm the potential of NADESs for extraction of bioactive constituents of medicinal plants and further suggest that NADES can improve the effects of bioactive extracts. Further studies are needed to clarify the influence of the studied NADES on the bioactivity of dissolved substances, and the possibility to use such extracts in the pharmaceutical and food industry.

The Verbascum species are widely used traditional herb remedies against respiratory, inflammatory conditions and disorders. In the present study methanol extract of the aerial parts of the endemic Verbascum nobile Velen, was investigated and two novel iridoid glycosides 1 and 2, together with nine known constituents: iridoids, phenylethanoids, and saponins characteristic of Verbascum genus were identified. Further, the biological activity of the extract and selected isolated compounds on concanavalin (Con A)-induced T cell proliferation and activation of human Jurkat T cell line and splenic murine CD3 T cells was evaluated. T cell growth was studied by colorimetric-based WST proliferation assay while DNA content, cell cycling, dynamic of cell proliferation, expression of activation markers, intracellular expression of cytokine IFN-γ, and phosphorylation of ERK were analyzed by flow cytometry. Caspase-mediated apoptosis resulting in a poly (ADP-ribose) polymerase (PARP) cleavage was assessed by colorimetric in-cell kit. It was found that the extract, and all tested compounds (1, 2, 3 and 9) inhibited lectin-induced cell growth of Jurkat T cell line. The novel compounds decreased the frequencies of cells in S phase without causing a significant cell cycle arrest at G1 phase, caspases-mediated apoptosis and/or a profound change in the dynamic of splenic murine CD3+ T cell proliferation. Both compounds showed stronger inhibitory effect on Con A-induced ERK phosphorylation than the known bioactive compounds 3 and 9, and suppressed the expression of early activation marker CD69, the intracellular level of IFN-γ, and the generation of CD3+IFN-γ+ effectors. Our data suggest that the novel iridoid glycosides might have a potential to modulate T cell-related pathologies.

The dichloromethane (DCM) extract of the fruits of Peucedanwn schottii Besser. ex DC. (Apiaceae) was subjected to high-performance counter-current chromatography (HPCCC) for the efficient and fast separation (30 min) and isolation of cimifugin using an ethyl acetate: water (1:1 v/v, K= 1.01) system. The analytical scale-optimized separation was easily scaled to semi-preparative conditions. Cimifugin (11.25% yield, 96.5% purity) was isolated for the first time from P. schottii and characterized by NMR spectroscopy. Cimifugin and the crude DCM extract were evaluated using ELISA microtiter assays for their inhibitory potential against the cholinesterases (acetylcholinesterase - AChE and butyrylcholinesterase - BChE), and tyrosinase (TYR), which are key enzymes for the treatment of some neurodegenerative diseases, i.e. Alzheimer's and Parkinson's. The crude extract exhibited a weak inhibitory activity against-AChE, BChE, and TYR (4.2, 35.5, and 0% at 100 μg mL-1 and 10.3, 40.0, and 12.2% at 200 μg mL-1, respectively), while cimifugin displayed low to moderate inhibition towards AChE and BChE (3.1 and 21.6%, respectively) at 200 μg mL'.

Plants from the Veronica genus are used across the world as traditional remedies. In the present study, extracts from the aerial part of the scarcely investigated Veronica austriaca L., collected from two habitats in Bulgaria&mdash, the Balkan Mountains (Vau-1) and the Rhodopi Mountains (Vau-2), were analyzed by nuclear magnetic resonance (NMR) spectroscopy. The secondary metabolite, arbutin, was identified as a major constituent in both extracts, and further quantified by high-performance liquid chromatography (HPLC), while catalpol, aucubin and verbascoside were detected at lower amounts. The effect of the extracts and of pure arbutin on the survival of neutrophils isolated from murine bone marrow (BM) were determined by colorimetric assay. The production of cytokines&mdash, tumor necrosis factor (TNF)-&alpha, and interferon (IFN)-&gamma, was evaluated by flowcytometry. While Vau-1 inhibited neutrophil vitality in a dose-dependent manner, arbutin stimulated the survival of neutrophils at lower concentrations, and inhibited cell density at higher concentrations. The Vau-1 increased the level of intracellular TNF-&alpha, while Vau-2 and arbutin failed to do so, and expanded the frequency of mature double TNF-&alpha, +/IFN-&gamma, hi neutrophils within the BM pool.

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.

A new coumarin-secoiridoid diglucoside, named Isofraxisecoside, was isolated from the stem bark of Fraxinus xanthoxyloides (G. Don) Wall. ex A. DC. along with nine known compounds. The structure of new compound has been determined on the basis of 1H, 13C, 2D NMR and HRMS methods.

Verbascum nigrum L. (dark mullein) is known to be a rich source of bioactive secondary metabolites, including high value iridoid and phenylethanoid glycosides, hence the development of alternative approaches for their sustainable supply is highly relevant. Here we report the induction of a transformed root culture of V. nigrum by applying sonication assisted Agrobacterium rhizogenes-mediated genetic transformation. Further, nuclear magnetic resonance (NMR)-based metabolomics approach has been successfully applied to study metabolic differences of the V. nigrum mother plant and related transformed roots. 1H NMR fingerprinting in combination with 2D NMR and multivariate data analysis revealed that the most abundant molecule in the hairy root culture is glutamine, which is not found in the mother plant tissue. These findings provide important insights on the metabolic alterations as a result of the transformation of the host plant genome and development of so-called hairy roots.

Forty-two samples of Sideritis species (Sideritis scardica, Sideritis raeseri, Sideritis syriaca, Sideritis taurica and Sideritis lanata) from the Balkan Peninsula were evaluated for their polyphenolic profiles in order to establish a correlation between the taxonomy, geographical location and nature and content of phenolic compounds. Eight compounds were detected in all analyzed Sideritis samples: 5-caffeoylquinic acid, lavandulifolioside, verbascoside, isoscutellarein 7-O-allosyl(1→2)glucoside, hypolaetin 7-O-[6‴-O-acetyl]-allosyl(1→2)glucoside, isoscutellarein 7-O-[6‴-O-acetyl]-allosyl(1→2)glucoside, 3′-O-methylhypolaetin 7-O-[6‴-O-acetyl]-allosyl(1→2)glucoside and 4′-O-methylhypolaetin 7-O-[6‴-O-acetyl]-allosyl-(1→2)-[6″-O-acetyl]-glucoside). They present from 50 to 80% of total phenolic content in S. scardica, S. raeseri, S. syriaca and S. taurica and up to 90% in S. lanata and the similarity of their polyphenolic profiles implies that they are systematically very closely related. Based on their polyphenolic patterns, very good differentiation between the samples of S. lanata (sect. Hesiodia) and S. scardica, S. syriaca and S. raeseri (sect. Empedoclia) was observed, that is mainly due to the very high content of 5-cafeoylquinic acid and very low amount of phenylethanoid glycosides and flavonoid glycosides. The obtained results demonstrate considerable degree of similarity between S. scardica, S. raeseri and Bulgarian S. syriaca that give contribution to the dilemma that Bulgarian S. syriaca is very similar to Turkish S. taurica and suggest further verification of its taxonomic status.

Sambucus plants have prominent place in folk medicine of the people from Europe and the Middle East. Sambucus ebulus (both above and under ground parts) preparations showed anti-neoplastic, antimicrobial (incl. antiviral) and anti-inflammatory properties. Elderberries accumulate sugars and fibers, vitamins and minerals, besides abundant secondary metabolites, as flavonoids, anthocyanins, phytosterols, triterpenes and iridoid glycosides, among others. Sambucus plants, however, also accumulate cyanogenic glycosides, whose presence is undesirable. Despite many applications so far the knowledge of the metabolites, accumulated in Sambucus species, is still limited and based mainly on determination of the major compounds. Here we report the application of 1H NMR metabolic fingerprinting in combination with principal component and hierarchical clustering analyses to reveal the metabolic differences of Sambucus mature and immature fruits, and plant leaves. Moreover, we show that immature fruits and leaves of S. ebulus have similar metabolome, which apparently undergoes significant changes during the fruit ripening stage. Sambunigrin was not detectable in any sample. To the best of our knowledge this is the first report on the systematic analysis of S. ebulus metabolome.

Background: Sideritis scardica and S. raeseri are very popular medicinal plants in Albania, Macedonia, and Bulgaria. Objective: Comparison of the phenolic, flavonoid, and diterpene contents and detailed chemical profiles, as well as data on antioxidant activity, comprising plant material from different locations in the Balkan Peninsula. Materials and Methods: Total phenolic and flavonoid contents for the studied Sideritis samples were determined spectrophotometrically. By HPLC-DAD-ESI-MSn analysis, 48 individual phenolic constituents were completely or tentatively identified. Chemical profiling of Sideritis major diterpenes and their quantification was performed by HPLC-ESI-MSD TOF analysis. A combination of two methods (ORAC and DPPH) was applied for evaluation of the antioxidant capacity of Sideritis extracts. Results: The results obtained demonstrated that Sideritis samples (S. scardica and S. raeseri) collected from Albania, Bulgaria and Macedonia are similar in their chemical profiles (they all contain phenylethanoid glycosides, flavonoid 7-O-diglycosides and acetylated flavonoid 7-Odiglycosides, and diterpenes: main components siderol and sideridiol); and antioxidant activity. Although some differences exist, they are not essential. Conclusion: This fact is a good basis for the formulation of range for parameters for quality control of Sideritis extracts and plant material. The studied cultivated plant material is similar in its chemical composition, profile and antioxidant activity to the wild growing samples.

Dwarf elder ( Sambucus ebulus L.) is a popular medicinal plant, used for centuries in the folk medicine of the Balkan Peninsula. S. ebulus preparations have shown anti-inflammatory, anti-neoplastic and antimicrobial properties, besides abundant wound healing, antioxidant and anti-ulcerogenic activities. We developed a scheme for isolation of individual compounds utilizing different chromatographic techniques, while the structure elucidation was performed by means of 1D and 2D NMR. Five flavonoid glycosides, e.g. quercetin-3- O -laminaribioside [ 1 ], isorhamnetin-3- O -laminaribioside [ 2 ], quercetin-3- O -rutinoside [ 3 ], isorhamnetin-3- O -rutinoside [ 4 ], isorhamnetin-3- O -glucoside [ 5 ], were identified accordingly. Compounds 1 and 2 are reported for the first time in the genus Sambucus . Several triterpenes – ursolic, oleanolic and maslinic acid – were tentatively identified by GC–MS. The evaluation of anti-herpes simplex virus type 1 and antioxidant (in ORAC FL and HORAC FL ) properties suggests that the dwarf elder fruits might serve as a powerful source of valuable molecules for various purposes.

Two new minor “Valeriana type” iridoid glycosides (1) and (2) along with 3 known flavonol glycosides [quercetin-3-O-β-glucopyranosyl-7-O-α-rhamnopyranoside (3), quercetin-3-O-β-glucopyranoside (4) and isorhamnetin-3-O-β-glucopyranoside (5)] were isolated from Sambucus ebulus L. leaves. Their structures were unambiguously elucidated by means of 1D- and 2D-NMR, and UPLC-TOF MS. Compound 2 is a rare representative of iridoid diglycosides, containing uncommon ribohexo-3-ulopyranosyl sugar moiety.

Harpagoside is an iridoid glycoside that was first isolated from Harpagophytum procumbens (devil's claw, Pedaliaceae), a medicinal plant in which it is the major constituent of the iridoid pool. Both the pure compound and devil's claw extracts have potent anti-rheumatic, anti-inflammatory and analgesic effects. According to the European Pharmacopoeia commercial devil's claw products should contain at least 1.2% harpagoside. However, the compound has also been isolated from several other plant species and in vitro plant culture systems. Recent advances in knowledge of harpagoside distribution, biosynthesis/accumulation and pharmacology are summarized in this review. We also discuss the possible synergism and/or antagonism between major constituents in harpagoside-containing phytopharmaceutical products. Finally, future perspectives for its potential application are highlighted.

The dichloromethane (DCM) extract of the fruits of Peucedanum schottii Besser ex DC. (Apiaceae) was subjected to high-performance counter-current chromatography (HPCCC) for the efficient and fast separation (30 min) and isolation of cimifugin using an ethyl acetate: water (1:1 v/v, K = 1.01) system. The analytical scale-optimized separation was easily scaled to semi-preparative conditions. Cimifugin (11.25% yield, 96.5% purity) was isolated for the first time from P. schottii and characterized by NMR spectroscopy. Cimifugin and the crude DCM extract were evaluated using ELISA microtiter assays for their inhibitory potential against the cholinesterases (acetylcholinesterase - AChE and butyrylcholinesterase - BChE), and tyrosinase (TYR), which are key enzymes for the treatment of some neurodegenerative diseases, i.e. Alzheimer's and Parkinson's. The crude extract exhibited a weak inhibitory activity against AChE, BChE, and TYR (4.2, 35.5, and 0% at 100 μg mL−1 and 10.3, 40.0, and 12.2% at 200 μg mL−1, respectively), while cimifugin displayed low to moderate inhibition towards AChE and BChE (3.1 and 21.6%, respectively) at 200 μg mL−1.

To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.

Verbascum xanthophoeniceum Griseb. is an endemic plant of the Balkan region, a representative of the genus Verbascum used in traditional medicine for respiratory and inflammatory disorders. The objective of this study was to evaluate in vivo and in vitro the antiinflammatory action of crude extract, different fractions and pure compounds obtained from V. xanthophoeniceum Griseb. Bioactive metabolites were isolated by the use of low-pressure chromatographic separation. Crude methanol extract (CME) was applied in a model of paw oedema and different fractions and substances were tested in vitro for their effect on NO and cytokine production by peritoneal macrophages, and on the COX-1 and COX-2 expression. The CME exerted inhibition on cobra venom factor (CVF)-induced oedema in mice, in correlation with reduced alternative pathway (AP) complement activity. A highly suppressive effect was expressed by nigroside VI on IL-6 and NO production and by forsythoside B on TNF-α production. Leucosceptoside B lowered NO release and COX-1 expression in macrophages. Verbascum xanthophoeniceum could serve as a promising source of active compounds with antiinflammatory action, particularly in complement-mediated disorders.

In this study extraction of polyphenols and flavonoids from cultivated hybrid Sideritis scardica × Sideritis syriaca, known for its rich content of phenolics and flavonoids with antioxidant activity, was investigated. Extractions have been done by ethanol and water–ethanol, respectively. High equilibrium values of the extracted species were obtained—17.55 mg/(g solid) total phenolics and 5.7 mg/(g solid) total flavonoids with ethanol as solvent. The influence of the solvent on the total yield and the content of biologically active compounds were studied. Maximum polyphenolics and flavonoids extraction was observed for water–ethanol solvent ratio 20/80. Increase of the content of ethanol in the solvents led to lower total yield of extracts but higher percentage of polyphenolics. The extraction kinetics showed that 90% of the phenolic compounds were extracted during the first 2.5 h. The experimental kinetics was described by a constant effective diffusion coefficient De = 1.5 × 10 −12 m 2 /s in the solid, accounting for the actual particle size distribution.

A previous report showed that extracts of cell suspension and transformed root cultures of Harpagophytum procumbens (commonly known as Devil's claw), an African plant with high medicinal value, exhibit strong antiinflammatory characteristics. The present work tests the ability of extracts, phenylethanoid-containing fractions and the major phenylethanoid glycoside isolated from the Devil's claw cultures, to inhibit acetylcholinesterase and butyrylcholinesterase, and the antioxidant activity in iron-related systems (e.g. ferric-reducing antioxidant power and ferrous ion-chelating capacity). The results indicated that the phenylethanoid fractions may be attractive for various commercial purposes since they displayed significant cholinesterase inhibitory activity (even higher than that of pure galanthamine in the case of butyrylcholinesterase inhibition assay). Crude methanolic extracts from cell and hairy root cultures of Devil's claw exhibited strong ferrous ion-chelating capacity (1.5-2 times higher than pure butylated hydroxyanisole, used as positive standard).

Cell suspension and Agrobacterium rhizogenes-transformed hairy root cultures of Devil’s claw (Harpagophytum procumbens), an African plant with high medicinal value, were cultivated in shake-flasks. A purification scheme to isolate their main active constituents (the phenylethanoid glycosides verbascoside, leucosceptoside A, β-OH-verbascoside and martynoside; structurally identified by NMR and LC–MS) was then developed, and their concentrations in the cultures were determined by UV spectrometry following HPLC separation. Preparations, extracts and the isolated phenylethanoid glycosides from the H. procumbens in vitro systems were tested on isolated murine macrophages to study their effects on nitric oxide (NO) and cytokine (TNF-α, IL-6) release and the expression of COX-1 and COX-2. They were also added to human serum to investigate their effects on the classical pathway of complement activation. The results indicate that the extracts and preparations of the in vitro systems, and pure verbascoside (their main active constituent), had strong anti-inflammatory properties, comparable to or even higher than that of pure harpagoside (a major anti-inflammatory constituent of intact Devil’s claw tubers). Thus, they have potential as new anti-inflammatory agents.

A cell suspension culture of Devil’s claw (Harpagophytum procumbens), a South African plant with high medicinal value, cultivated under submerged conditions showed stable growth and accumulated high amounts of biomass (18.2 g l−1). Flow cytometry analyses of the suspension’s cell cycle kinetics showed that proportions of cells in G0/G1 and S phases varied insignificantly (between 69–76% and 9–13%, respectively) during the cultivation, while the proportion of G2/M-phase cells increased until day 8 of cultivation, when the exponential phase of cell growth ended. Metabolite production in the culture was studied through simultaneous determination of three bioactive phenylethanoid glycosides (verbascoside, β-OH-verbascoside and leucosceptoside A) by high performance liquid chromatography. It was found that suspended Devil’s claw cells accumulated mainly verbascoside (517.3 mg l−1), followed by leucosceptoside A (107.1 mg l−1) and β-OH-verbascoside (80.3 mg l−1). In addition, several fatty acids and β-sitosterol were identified in the cell suspension by gas chromatographic-mass spectrometry analysis. Comparison of the results with previously acquired data for Harpagophytum procumbens transformed roots indicate that cell suspensions cultures are more promising as potential commercial sources of metabolites such as phenylethanoid glycosides.

A cell suspension culture of devil’s claw (Harpagophytum procumbens), an African plant with high medicinal value, was induced from previously obtained callus cultures, cultivated in shake-flasks, and a purification scheme to isolate the main active constituents that accumulated in the biomass was developed. The antioxidant activities of total methanol extracts, collected fractions (phenylethanoids, terpenoids and sugars) and specific active constituents (β-OH-verbascoside, verbascoside and leucosceptoside A) were then evaluated in 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and superoxide anion ( O 2 - ) radical scavenging and oxygen radical absorbance capacity (ORACFL) assays. The most active compounds were found to be β-OH-verbascoside (in DPPH· and superoxide radical scavenging assays) and leucosceptoside A (in ORAC assays). The phenylethanoid fraction may be attractive for various commercial purposes since it displayed significant antioxidant activity and it can be conveniently and economically prepared by a single-step column separation of extracts.

Harpagophytum procumbens is an herbaceous plant with high medicinal value that grows in the Kalahari Desert region of Africa. Devil's claw plant tubers have been used since time immemorial by the n...

An LC–ESI-MS analysis was performed to determine the iridoid composition of Lamium album , Lamium amplexicaule , Lamium garganicum , Lamium maculatum , and Lamium purpureum . Nine iridoids known to occur in these species, lamalbid, sesamoside, lamiol, 5-deoxylamiol, shanzhiside methyl ester, caryoptoside, penstemoside, lamioside, and barlerin, could be detected. Confident identification of the individual compounds was achieved by a combination of HPLC retention times, both positive and negative ionization modes in MS and the presence of cluster and fragment ions. Iridoid glucosides new to four of the studied species were reported here for the first time. In most cases, the observed iridoid profiles were consistent and only small intraspecific variations were detected. Some chemosystematic implications from the observed iridoid composition were discussed.

The resurrection plant Haberlea rhodopensis was used to study dynamics of drought response of photosynthetic machinery parallel with changes in primary metabolism. A relation between leaf water content and photosynthetic performance was established, enabling us to perform a non-destructive evaluation of the plant water status during stress. Spectroscopic analysis of photosynthesis indicated that, at variance with linear electron flow involving photosystem (PS) I and II, cyclic electron flow around PSI remains active till almost full dry state at the expense of the linear electron flow, due to the changed protein organization of photosynthetic apparatus. We suggest that, this activity could have a photoprotective role and prevent a complete drop in adenosine triphosphate (ATP), in the absence of linear electron flow, to fuel specific energy-dependent processes necessary for the survival of the plant, during the late states of desiccation. The NMR fingerprint show significant metabolic changes in several pathways. Due to the declining of linear electron flow accompanied by biosynthetic reactions during desiccation, a reduction of the ATP pool during drought was observed, which was fully and quickly recovered after plants rehydration. We found a decline of valine accompanied by lipid degradation during stress, likely to provide alternative carbon sources for sucrose accumulation at late stages of desiccation. This accumulation, as well as the increased levels of glycerophosphodiesters during drought stress could provide osmoprotection to the cells.

1. Subject and sourceLamium album L. is a perennial herb used in folk medicine as blood tonic,antispasmodic and anti-inflammatory agent (Bremness, 1995). Lamium maculatumL. has been used in the Chinese folk medicine for treatment of trauma, fracture andhypertension (Shuya et al., 2003). The plant materials of L. album and L. maculatumwere collected in May 2003 at Vlado Trichkov region and Losen Mountain, Bulgariaand identified by Dr. Ljuba Evstatieva. The voucher specimens (Co 1039 and 1038)

Five C10 iridoid glycosides (shanzhiside methyl ester, lamalbide, lamiide, sesamoside, and 5‐desoxysesamoside) were examined by electrospray ionization mass spectrometry (ESI‐MS). Considerable differences were observed in positive ion and negative ion mode. Only the positive ion spectra were useful for molecular mass determination on the basis of molecular alkali metal adducts, with the exception of two iridoids containing all 5β‐OH, 7β‐OH and 8β‐OH groups. Fragment characteristics for distinct functionalities were observed. Chelation studies of each iridoid with three different alkali metals were also conducted.

The aerial parts of Carthamus lanatus (Asteraceae) afforded four new oxygenated bisabolane fucosides, 10-hydroperoxy-bisabola-2,11-diene 7-O-beta-D-fucopyranoside, 11-hydro-peroxy-bisabola-2,9-diene 7-O-beta-D-fucopyranoside, 10-hydroxy-bisabola-2,11-diene 7-O-beta-D-fucopyranoside and 11-hydroxy-bisabola-2,9-diene 7-O-beta-D-fucopyranoside together with the known compounds a-bisabolol beta-D-fucopyranoside, asperuloside, sitosterol 3-O-beta-D-glucoside and stigmasterol 3-O-beta-D-glucoside. Asperuloside appears to be the second representative of the iridoid monoterpene group found in the plant family Asteraceae, which until recently was considered to lack iridoids. The main constituent a-bisabolol fucoside exhibited noticeable antibacterial and cytotoxic activities.

The genus Verbascum (mulleins), belonging to the family Scrophulariaceae, comprises about 360 species of flowering plants. The leaves, flowers and whole aerial parts of Verbascum spp. have been widely used in traditional medicine for the treatment of respiratory and inflammatory disorders and also display powerful wound healing activity. Verbascum species are found to accumulate several groups of bioactive molecules, therefore they might be utilized as attractive sources of new (drug) leads. The present review attempts to provide an up-to-date comprehensive overview on phytochemical and pharmacological aspects of Verbacum spp. research along with some successful examples of growing (and transforming) mulleins in vitro.

Ethnopharmacological relevance Verbascum xanthophoeniceum is a mullein plant, typical of Balkan region and some parts of Turkey, traditionally used as phytotherapeutic agent due to its anti-inflammatory properties. It is rich in phenylethanoid and iridoid metabolites whose anti-inflammatory properties are under characterization. Materials and methods The role of Verbascum xanthophoeniceum crude methanolic extract and its isolated phenylethanoid glycoside verbascoside have been evaluated, in comparison to a saw palmetto extract, on a human in vitro model of androgen-regulated prostate epithelium, the LNCaP cell line. Cytotoxicity and DHT-induced free and total PSA secretion have been thoroughly studied. Results We have found that similar to saw palmetto, Verbascum xanthophoeniceum extract and its isolated phenylethanoid glycoside verbascoside have no cytotoxicity in human LNCaP prostate epithelial cells, whereas an inhibitory effect on the DHT-induced free and total PSA secretion, a recognized anti-androgen like activity, has been shown in case of both Verbascum xanthophoeniceum extract and pure verbascoside. Furthermore, in the absence of the endogenous androgen DHT, an androgen-like activity in Verbascum xanthophoeniceum is detectable as it is for saw palmetto, suggesting that a mixed androgen-antiandrogen activity is present. Conclusions For the first time, Serenoa repens and Verbascum xanthophoeniceum extracts have shown an absence of cytotoxicity and an inhibitory effect on DHT-induced PSA secretion in an in vitro model of human prostate epithelium, whereas the phenylethanoid glycoside verbascoside appeared to explain only part of the Verbascum xanthophoeniceum inhibitory activity on PSA secretion.

A new iridoid diglucoside, 10- O -( E )- p -coumaroylmelittoside ( 1 ) and a new flavone glucoside, O -(6″- O -Acetyl-)-β-glucopyranosylchrysoeriol ( 2 ), together with four known flavone glycosides, were isolated from the aerial parts of Sideritis lanata . The structures of the new compounds were elucidated by spectroscopic methods, 2D NMR and MS.

Ethnopharmacological relevance Verbascum xanthophoeniceum is a representative of mullein species with a strong tradition of use in folk medicine as a remedy in inflammatory and infectious contexts. This plant accumulates phenylethanoid and iridoid metabolites with a partially characterized bioactivity. Materials and methods Here, we compared anti-inflammatory effects of Verbascum xanthophoeniceum crude extract, its fractions, isolated iridoid glycosides, including aucubin, ajugol, harpagide, harpagoside, nigroside III and nigroside VI, and phenylethanoid glycosides verbascoside and forsythoside B in primary cultures of normal human keratinocytes (NHK). The gene expression, synthesis, and release of soluble pro-inflammatory chemokines, such as IL-8, MCP-1 and IP-10, in dormant and IFN-γ−activated NHK were investigated, and IC 50 for each extract/individual substance was determined. Results We found that the phenylethanoid glycosides verbascoside and forsythoside B were effective, dose-dependent inhibitors of gene expression and de novo synthesis of all the chemokines, whereas the iridoid glycosides and phenylpropanoid aglycone rosmarinic acid displayed a poor and selective inhibition. Accordingly, the fraction of the crude extract containing verbascoside effectively impaired both spontaneous and induced chemokine expression in NHK. Conclusion This is the first report on the identification of active constituents of Verbascum xanthophoeniceum possessing anti-inflammatory properties towards human keratinocytes. Phenylethanoid glycosides exerted exquisite corticosteroid-like inhibition of pro-inflammatory chemokines at transcriptional and translational levels.

The genus Verbascum L. (mulleins) comprises of about 360 species of flowering plants in the Scrophulariaceae family. Mulleins have been used in the traditional folk medicine for centuries, for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. Here we report the application of 1H NMR metabolic fingerprinting in combination with principal component analyses (PCA) in five different Verbascum species. Based on the obtained results mulleins were divided in two groups: group A (Verbascum phlomoides and Verbascum densiflorum) and group B (Verbascum xanthophoeniceum, Verbascum nigrum and Verbascum phoeniceum). Further it was found that the plants in group B accumulate higher amounts of bioactive iridoid and phenylethanoid glycosides. V. xanthophoeniceum and V. nigrum accumulate higher amounts of the pharmaceutically-important harpagoside (∼0.5% on dry weight basis) and verbascoside, forsythoside B and leucosceptoside B (in total 5.6-5.8% on dry weight basis), which underlines the possibility for their application in pharmaceutical industry. To the best of our knowledge this is the first report on the analyses of Verbascum sp. leaf metabolome.