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Comfrey (Symphytum officinale L., Boraginaceae) root preparations are used as both traditional remedies and therapeutic agents in treating pain and inflammation associated with joint, bone, and muscle ailments. Even though numerous phytochemicals contribute to the beneficial effects of comfrey, the presence of toxic pyrrolizidine alkaloids (PAs) overshadows its uses.In this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation.The phytochemical profiling was performed by LC-HRMS/MS. The quantification of PAs and major phenolic compounds was carried out by LC-MS/MS and LC-DAD. Antioxidant and enzyme inhibitory activity was determined by in vitro free radical scavenging, ion reducing, metal chelating, cholinesterase, tyrosinase, amylase, and glucosidase assays. Using an ex vivo model of LPS-stimulated neutrophils, their viability (as measured by flow cytometry) and the release of IL-1β, IL-8, and TNF-α were determined (ELISA assay).12 phenolic acids, six PAs, three organic acids, two fatty acids, and two sugars were identified in the obtained comfrey extracts. The PA-depleted materials contained PAs levels below 2 ppm, whereas the removal of mucilage increased the content of rosmarinic acid, globoidnan A, globoidnan B, and rabdosiin. PA-depletion did not significantly affect the antioxidant potential. However, the radical scavenging and metal reducing properties were higher in the mucilage-depleted extracts. Neither PA-depletion nor mucilage-depletion had considerable effects on the in vitro inhibitory activity of cholinesterases, tyrosinase, amylase, and glucosidase or release of ex vivo pro-inflammatory cytokines (e.g., IL-1β, IL-8, and TNF-α) in LPS-stimulated neutrophils.In light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

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Targeting pancreatic lipase and α-amylase by digestion-derived fractions of ethanolic-aqueous (60%

Sambucus nigra (elderberry) leaves were used in folk medicine to treat skin inflammations, ulcers, burns or boils, as well as to treat wounds, including infected and chronic ones. For centuries, elderberry leaves have been used mainly in eastern and southern Europe, as well as in western Asia.The study aimed to investigate the anti-inflammatory and antioxidant activity of four different extracts, such as aqueous and ethanolic prepared at room temperature and the solvent's boiling point, from the leaves of elderberry.The effect of extracts both on the secretion of cytokines (TNF-α, IL-1β, and IL-8) and reactive oxygen species (ROS) by neutrophils stimulated with bacteria-derived products was investigated. The cytotoxicity of extracts was analyzed by staining with propidium iodide measured by flow cytometry. The anti-inflammatory activity of extracts was also investigated through their influence on lipoxygenase activity. The antioxidant properties, including scavenging superoxide anion, hydrogen peroxide, nitric oxide, and 2,2-diphenyl-1-picrylhydrazyl radical were investigated in cell-free systems. The total content of phenolic compounds was tested using the Folin-Ciocalteu reagent. The qualitative and quantitative determination of the content of individual phenolic acids and flavonoids was performed by HPLC-DAD-MSElderberry leaves extracts turned out to affect the inflammatory response of neutrophils by inhibiting the secretion of TNF-α and ROS. The ethanolic and aqueous extracts at a concentration of 50 μg × mLOur research has confirmed that elderberry leaves are a plant material with anti-inflammatory activity, especially against reactive oxygen species, and a potentially rich source of antioxidants. Preliminary analyses performed in this study could be the first step in confirming the traditional use of elderberry leaves in relieving inflammation.

The Artemisia L. genus includes over five hundred species with great economic and medicinal properties. Our study aimed to provide a comprehensive metabolite and bioactivity profile of Artemisia campestris subsp. lednicensis (Spreng.) Greuter & Raab-Straube collected from north-eastern Romania. Liquid chromatography with tandem high-resolution mass spectrometry (LC-HRMS/MS) analysis of different polarity extracts obtained from the aerial parts led to the identification of twelve flavonoids, three phenolic acids, two sesquiterpene lactones, two fatty acids, one coumarin, and one lignan. The antioxidant and enzyme inhibitory properties were shown in the DPPH (0.71–213.68 mg TE/g) and ABTS (20.57–356.35 mg TE/g) radical scavenging, CUPRAC (38.56–311.21 mg TE/g), FRAP (121.68–202.34 mg TE/g), chelating (12.88–22.25 mg EDTAE/g), phosphomolybdenum (0.92–2.11 mmol TE/g), anti-acetylcholinesterase (0.15–3.64 mg GALAE/g), anti-butyrylcholinesterase (0–3.18 mg GALAE/g), anti-amylase (0.05–0.38 mmol ACAE/g), anti-glucosidase (0.43–2.21 mmol ACAE/g), and anti-tyrosinase (18.62–48.60 mg KAE/g) assays. At 100 μg/mL, Artemisia extracts downregulated the secretion of tumor necrosis factor (TNF)-α in a lipopolysaccharide (LPS)-stimulated human neutrophil model (29.05–53.08% of LPS+ control). Finally, the Artemisia samples showed moderate to weak activity (minimum inhibitory concentration (MIC) > 625 mg/L) against the seventeen tested microbial strains (bacteria, yeasts, and dermatophytes). Overall, our study shows that A. campestris subsp. lednicensis is a promising source of bioactives with putative use as food, pharmaceutical and cosmetic ingredients.

Dermatophyte infections represent an important public health concern, affecting up to 25% of the world’s population. Trichophyton rubrum and T. mentagrophytes are the predominant dermatophytes in cutaneous infections, with a prevalence accounting for 70% of dermatophytoses. Although terbinafine represents the preferred treatment, its clinical use is hampered by side effects, drug–drug interactions, and the emergence of resistant clinical isolates. Combination therapy, associating terbinafine and essential oils (EOs), represents a promising strategy in the treatment of dermatophytosis. In this study, we screened the potential of selected Apiaceae EOs (ajowan, coriander, caraway, and anise) to improve the antifungal activity of terbinafine against T. rubrum ATCC 28188 and T. mentagrophytes ATCC 9533. The chemical profile of EOs was analyzed by gas chromatography. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of EOs/main compounds were determined according to EUCAST-AFST guidelines, with minor modifications. The checkerboard microtiter method was used to identify putative synergistic combinations of EOs/main constituents with terbinafine. The influence of EOs on the viability and pro-inflammatory cytokine production (IL-1β, IL-8 and TNF-α) was determined using an ex vivo human neutrophils model. The binary associations of tested EOs with terbinafine were found to be synergistic against T. rubrum, with FICI values of 0.26–0.31. At the tested concentrations (6.25–25 mg/L), EOs did not exert cytotoxic effects towards human neutrophils. Anise EO was the most potent inhibitor of IL-1β release (46.49% inhibition at 25 mg/L), while coriander EO displayed the highest inhibition towards IL-8 and TNF-α production (54.15% and 54.91%, respectively). In conclusion, the synergistic combinations of terbinafine and investigated Apiaceae EOs could be a starting point in the development of novel topical therapies against T. rubrum-related dermatophytosis.

The purpose of this study was to analyze the HPLC-DAD-ESI-Q-TOF-MS/MS profile of the methanolic extract obtained from the aerial parts of Verbascum blattaria L. and develop efficient high-performance countercurrent chromatography (HPCCC) methods for isolation of its major iridoid glycosides. Additionally, the potential anti-inflammatory activities of the extract and two of the isolated compounds were evaluated using a human neutrophil model. LC–MS analysis led to the identification of 35 constituents, of which 18 iridoids, eight saponins, three phenylethanoids, four flavonoids and two phenolic acids. Scropolioside F (A, 2 mg), scrophuloside A3 (B, 16 mg) and gmelinoside L (C, 13 mg) were obtained from 300 mg of the crude methanolic extract by HPCCC with the help of two different biphasic solvent systems: n-hexane/ethyl acetate/n-butanol/water (1/2/1/2; v/v/v/v) and n-hexane/ethyl acetate/methanol/water (1/19/1/19; v/v/v/v). The crude methanolic extract (100 μg/mL) down-regulated lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) release in a comparable extent to dexamethasone (25 μM). Gmelinoside L (50 μM) was more active than scrophuloside A3, producing inhibition percentages of 43.52% and 41.28% on interleukin 8 (IL-8) and TNF-α level, respectively. The HPCCC methods developed in the current work may serve to large scale isolation of iridoid glycosides from Verbascum species or other iridoid abundant-genera.

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.

Less-common fruits from Cornus spp. (Cornaceae), also named dogwoods, have shown antidiabetic, antibacterial and anti-allergic properties and are thus considered a source of phytochemicals that are beneficial to human health. The study aimed to compare the chemical compositions of the aqueous and ethanolic extracts of lyophilized fresh-picked and commercially available dried fruits of Cornus mas (Cm, cornelian cherry) and Cornus alba (Ca) fruits using HPLC-DAD-MS/MS method. Simultaneously, the α-amylase and pancreatic lipase (PL) inhibitory activities of the prepared extracts were compared by in vitro fluorescence assay based on the kinetic hydrolysis of starch or oleate ester of 4-methylumbelliferone (MUO), respectively. Additionally, a bio-assay guided identification of compounds potentially responsible for the inhibition of pancreatic enzymes was performed. Iridoids (loganic acid, cornuside) and anthocyanins (pelargonidin 3-O-galactoside) were identified in the Cm fruit extracts. Flavonoids, such as quercetin and kaempferol derivatives, were detected in the Ca fruit extracts. The chromatographic separation of the constituents of Ca fruit provided a fraction containing phenolic acids derivatives, which inhibited PL activity by 69.9 ± 4.5% at a concentration of 7.5 μg·mL−1. The IC50 of hydroxytyrosol glucoside, isolated from the most active Ca fraction, was 0.99 ± 0.10 mg·mL−1 indicating other constituents responsible for the fraction activity. The most active subfraction from Cm fruit (7.5 μg·mL−1), which inhibited PL activity by 28.3 ± 1.5%, contained pelargonidin 3-O-galactoside. Loganic acid and cornuside in highly pure form did not inhibit lipase activity. The phytochemical constituents of Cm, and particularly of Ca fruit extracts, can inhibit pancreatic enzymes and thus might be considered effective preparations in the prevention and control of hyperlipidemia related diseases. Keywords: Anthocyanins, Digestive enzymes, Iridoids, Metabolic syndrome, Phenolic compounds

A screening of inhibitory activity of α-amylase, as well as pancreatic lipase (PL), under the influence of aqueous and ethanolic preparations from 12 plant materials was performed. The most active aqueous extracts from the fruits of Chaenomeles japonica (CJ) and Hippophae rhamnoides (HR) were selected for artificial gastrointestinal digestion (GID). The aim of this study was to evaluate the inhibitory effect of the fractions obtained after GID on PL and α-amylase activities using a fluorescence assay. The changes in the composition of crude extracts in GID aliquots were followed by analysis with HPLC-DAD-MSn method in order to indicate active constituents. The main constituents of CJ and HR extracts were procyanidins and isorhamnetin derivatives, respectively. The most abundant compounds of extracts were found in all compartments of the digestion model correlated with relevant lipase/α-amylase inhibitory activity. What is more, the gastric and intestinal fractions inhibited enzymatic activity by at least 40%.

Due to certain differences in terms of molecular structure, isoquinoline alkaloids from Chelidonium majus engage in various biological activities. Apart from their well-documented antimicrobial potential, some phenanthridine and protoberberine derivatives as well as C. majus extract present with anti-inflammatory and cytotoxic effects. In this study, the LC&ndash, MS/MS method was used to determine alkaloids, phenolic acids, carboxylic acids, and hydroxybenzoic acids. We investigated five individually tested alkaloids (coptisine, berberine, chelidonine, chelerythrine, and sanguinarine) as well as C. majus root extract for their effect on the secretion of IL-1&beta, IL-8, and TNF-&alpha, in human polymorphonuclear leukocytes (neutrophils). Berberine, chelidonine, and chelerythrine significantly decreased the secretion of TNF-&alpha, in a concentration-dependent manner. Sanguinarine was found to be the most potent inhibitor of IL-1&beta, secretion. However, the overproduction of IL-8 and TNF-&alpha, and a high cytotoxicity for these compounds were observed. Coptisine was highly cytotoxic and slightly decreased the secretion of the studied cytokines. The extract (1.25&ndash, 12.5 &mu, g/mL) increased cytokine secretion in a concentration-dependent manner, but an increase in cytotoxicity was also noted. The alkaloids were active at very low concentrations (0.625&ndash, 2.5 &mu, M), but their potential cytotoxic effects, except for chelidonine and chelerythrine, should not be ignored.

Linden flower is a wildly used plant material among patients in the treatment of common cold symptoms and mucosa inflammations. However, the structure and bioactivity of flavan-3-ol derivatives present in infusions from flowers of Tilia cordata have not been studied so far. The aim of current study was to isolate and identify main procyanidins present in the flowers of small-leaved lime and to evaluate their influence on the inflammatory response of human neutrophils ex vivo. The chemical structure of isolated compounds was established by 1D and 2D NMR experiments. The bioactivity of obtained compounds was tested in human neutrophils model. Cytotoxicity and influence of compounds on apoptosis was established by flow cytometry. The levels of produced cytokines were established by ELISA after stimulation with lipopolysaccharide (LPS). The inhibition of the production of reactive oxygen species was checked by luminol-dependent chemiluminescence method after N-formylmethionyl-leucyl-phenylalanine (f-MLP) induction. The phytochemical work resulted in the isolation of 10 compounds. Compounds were identified as oligomeric procyanidins and their precursor epicatechin. The potential anti-inflammatory activity of compounds was evaluated in the concentration range 5–20 μM. All compounds were able to decrease the production of ROS from f-MLP-stimulated neutrophils. Most of compounds were able to inhibit the LPS-induced release of IL-8. Some trimeric and tetrameric derivatives were also able to decrease the production of MIP-1β. Obtained results partially support the traditional usage of infusion from lime flowers in the treatment of symptoms of inflammation and irritation of mucosa in common cold, pharyngitis and tonsillitis.

Dermatophyte infections represent a significant public health concern, with an alarming negative impact caused by unsuccessful therapeutic regimens. Natural products have been highlighted as a promising alternative, due to their long-standing traditional use and increasing scientific recognition. In this study, honokiol and magnolol, the main bioactives from Magnolia spp. bark, were investigated for their antidermatophytic activity. The antifungal screening was performed using dermatophyte standard strains and clinical isolates. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) were determined in accordance with EUCAST-AFST guidelines, with minor modifications. The effects on ergosterol biosynthesis were assessed in Trichophyton rubrum cells by HPLC-DAD. Putative interactions with terbinafine against T. rubrum were evaluated by the checkerboard method. Their impact on cells’ viability and pro-inflammatory cytokines (IL-1β, IL-8 and TNF-α) was shown using an ex vivo human neutrophils model. Honokiol and magnolol were highly active against tested dermatophytes, with MIC and MFC values of 8 and 16 mg/L, respectively. The mechanism of action involved the inhibition of ergosterol biosynthesis, with accumulation of squalene in T. rubrum cells. Synergy was assessed for binary mixtures of magnolol with terbinafine (FICI = 0.50), while honokiol-terbinafine combinations displayed only additive effects (FICI = 0.56). In addition, magnolol displayed inhibitory effects towards IL-1β, IL-8 and TNF-α released from lipopolysaccharide (LPS)-stimulated human neutrophils, while honokiol only decreased IL-1β secretion, compared to the untreated control. Overall, honokiol and magnolol acted as fungicidal agents against dermatophytes, with impairment of ergosterol biosynthesis.

Ethnopharmacological relevance Solidago virgaurea L. (also known as European goldenrod) is a pharmacopoeial plant material popularly used by patients in the form of an infusion. It was traditionally used in Europe and North America for the treatment of urinary tract conditions. It is also reported as a topical agent for skin disorders. Aim of the study Gut microbiota metabolism plays a crucial role in the bioavailability of natural products contained in plant extracts taken orally. The aim of the current study was to establish the biotransformation of compounds contained in an infusion from goldenrod using human and piglet fecal microbiota in vitro. The permeability of unmetabolized natural products and gut microbiota metabolites was evaluated using a Caco-2 cell model. Preliminary anti-inflammatory assays of raw extract using human neutrophils were also established. Material and methods An infusion was prepared from Solidaginis virgaureae herba commercially available on the market. The characterization of the raw extract was performed by UHPLC-DAD-MS method. The infusion was incubated with human or swine fecal samples in anaerobic conditions. Metabolism products were analyzed and identified by UHPLC-DAD-MS technique. The permeability of the natural products contained in the raw infusion and after metabolism was checked by UHPLC method. The influence of raw extracts on proinflammatory functions of human neutrophils after LPS stimulation was established by flow cytometry and ELISA. Results The experiments showed that goldenrod infusion contains mainly caffeoylquinic acid derivatives, flavonoids, and some phenylpropanoids. Natural products present in the extract were transformed by human and swine microbiota to smaller molecules mainly phenylpropanoid acid derivatives. The permeability assays showed that most of the parental compound present in the infusion cannot cross the gut epithelial barrier. In contrast, metabolites were able to cross the Caco-2 monolayer. Depending on the structure, different possible mechanisms of transport were observed. The infusion did not significantly influence the proinflammatory functions of human neutrophils. Conclusions Following oral administration of goldenrod infusion, phytochemicals are prone to undergoing metabolism by gut microbiota to smaller phenylpropionic acid derivatives that can be bioavailable after crossing the gut epithelial barrier to be further metabolized and distributed. Detected metabolites should be considered as potentially active compounds responsible for the bioactivity of the raw plant material in vivo.

Plants belonging to the family Oleaceae have been traditionally used in treatment of various inflammatory skin disorders. One of the most well-known species is Olea europaea L. (olive), cultivated in the Mediterranean countries. Another species is Ligustrum vulgare L. (common privet), occurring particularly in Northern Europe and Asia. The aim of the study was a comparison of the composition of aqueous and ethanolic extracts from leaves of O. europaea and L. vulgare (HPLC-DAD-MS), and determination of the total content of phenolics and flavonoids, as well as the content of the major compound, oleuropein. Secondly, we aimed to study the protective effect of extracts on reactive oxygen species (ROS) production by human fibroblast cells (NHDF), cell viability (MTT assay), and apoptosis rate (Annexin V/propidium iodide staining) after UVA-irradiation. The phytochemical analysis allowed us to identify compounds belonging to the groups of flavonoids, phenylpropanoids and secoiridoids in the extracts. The compounds from the group of lignans (olivil) were identified as being unique to O. europaea extracts. Echinacoside, ligustroflavone and ligustaloside A were identified in L. vulgare extracts in contrast to olive preparations. It was established that the aqueous and ethanolic extracts from leaves of both species, except the privet aqueous extract at a concentration of 5 μg/ml, did not show any significant inhibition of ROS production after UVA-irradiation in the model of NHDF cell line. The aqueous extracts of both species at concentrations of 5 and 25 μg/ml had a protective effect on the viability of UVA-treated cells in contrast to the ethanolic extract. In conclusion, no significant difference in the activity of olive and privet leaf extracts has been observed, which suggests that both plant materials’ extracts, particularly aqueous ones, are effective herbal medicines and photoprotectors, which – to some extent – confirms the use of their preparations in skin disorders.

Ethnopharmacological relevance During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1β and TNF-α) release and downregulation of adhesive molecules. Aim of the study During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. Materials and methods The effect on IL-8, IL-1β and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. Results We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of β2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1β release comparable with budesonide, used as a positive control. Conclusions The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I. helenium and G. squarrosa for a treatment of inflammation-based diseases in respiratory tract.

A wide range of plant-derived preparations have been used against skin inflammatory disorders and as wound healing agents in traditional medicine. The purpose of the study was to determine the antioxidant activity of aqueous and 70% ethanolic extracts from eleven species of plants traditionally used in Poland to treat inflammatory skin diseases. The ability of extracts to scavenge 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), hydrogen peroxide (H2O2), and superoxide anion (O2•−), was studied. In non-cellular studies, an analysis of the anti-inflammatory effect on the activity of enzymes, such as hyaluronidase (HYAL) and lipoxygenase (LOX), was also performed. The chemical profiles of the most active extracts were achieved by applying the UHPLC-DAD-MSn method, and the sum of polyphenols in all tested extracts was determined by the colorimetric method with the Folin–Ciocalteu reagent. The scope of the extracts’ influence on enzyme activity was significantly lower than their antioxidant activity. All extracts have shown high activity in free radical scavenging against DPPH. The ethanolic extracts have shown high potential to scavenge H2O2. The study of composition showed that the main components of the tested extracts were flavonoids, such as luteolin, apigenin, kaempferol, and quercetin derivatives, as well as caffeoylquinic acids, caffeic acid, and its conjugates.

Ethnopharmacological relevance Symphytum officinale L. (comfrey, Boraginaceae) has been traditionally used for millennia in joint distortions, myalgia, bone fractures and hematomas. However, key activity-determining constituents and molecular mechanisms underlying its use have not been completely elucidated. Aim of the study The objective of this study was to isolate and identify the major compounds from a hydroethanolic root extract of S. officinale and evaluate their antioxidant potential, alongside their effect on the cytokine production of ex vivo stimulated neutrophils, thus providing scientific support for the traditional use of comfrey root. Material and methods Four caffeic acid oligomers were isolated from comfrey roots by liquid-liquid chromatography, their structures being established by MS and NMR analyses. In vitro antioxidant evaluation was performed by DPPH and ABTS assays. The cytotoxicity of isolated compounds was established by flow cytometry. The effect on cytokine release, such as interleukin (IL)-1β, IL-8 and tumor necrosis factor alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated neutrophils was determined by enzyme-linked immunosorbent assay (ELISA). Results The main constituents found in comfrey root were represented by four caffeic acid oligomers, namely globoidnan B (1), rabdosiin (2), rosmarinic acid (3) and globoidnan A (4). Rabdosiin, globoidnans A and B were isolated for the first time from S. officinale. In the in vitro antioxidant tests, compound 2 was the most active, with EC50 values in DPPH and ABTS assays of 29.14 ± 0.43 and 11.13 ± 0.39, respectively. Neutrophils’ viability over the tested concentration domain of 12.5–50 μM was not altered. At 50 μM, all compounds significantly inhibited IL-1β release, with compound 3 (45.60% release vs. LPS stimulated neutrophils) being the most active, followed by compounds 1 (53.85%), 2 (69.89%) and 4 (60.68%). Conclusions The four caffeic acid oligomers reported in S. officinale root may contribute to the overall anti-inflammatory activity for which comfrey preparations are used in traditional medicine.

The fruits of some Cornus species (dogwoods) are used in traditional medicine and considered potential anti-diabetic and hypolipemic agents. The aim of the study was to determine the ability of extracts from Cornus alba (CA), Cornus florida (CF), and Cornus sanguinea (CS) to inhibit digestive enzymes namely &alpha, amylase, pancreatic lipase, and &alpha, glucosidase, as well as isolation of compounds from plant material with the strongest effect. In addition, the phytochemical profile and antioxidant activity of extracts from three dogwoods were compared with HPLC-DAD-MS/MS and DPPH scavenging assay, respectively. Among the aqueous-ethanolic extracts, the activity of &alpha, amylase was the most strongly inhibited by the fruit extract of CA (IC50 = 115.20 &plusmn, 14.31 &mu, g/mL) and the activity of &alpha, glucosidase by the fruit of CF (IC50 = 38.87 &plusmn, 2.65 &mu, g/mL). Some constituents of CA fruit extract, such as coumaroylquinic acid, kaempferol, and hydroxytyrosol derivatives, were isolated. Among the three species of dogwood studied, the greatest biological potential was demonstrated by CA extracts, which are sources of phenolic acids and flavonoid compounds. In contrast, iridoid compounds or flavonoid glycosides found in fruits of CF or CS extracts do not play a significant role in inhibiting digestive enzymes but exert antioxidant activity.

The traditional role of Lamium album L. (white dead nettle, Lamiaceae) in providing relief from pain in rheumatism as well as vaginal and cervical inflammation was described. The aim of the study was to screen for the anti-inflammatory bioactivity of compounds isolated from aqueous-methanolic extract of Lamium album herb in human neutrophils (PMNs). The effect of the compounds on the inhibition of selected inflammatory markers released by neutrophils, such as cytokines (IL-8, TNF-&alpha, ), was studied. The molecular masses and the purity of compounds were determined using high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MSn). The level of cytokines production after incubation with the compounds (1&ndash, 25 &micro, M) was measured by ELISA. Two derivatives of quercetin, not previously described, were isolated in this study. Phenylpropanoids (verbascoside and phlinoside D), as well as iridoids (lamalbid, and shanzhiside methyl ester), and flavonoids revealed to be more significant inhibitors of IL-8 secretion than TNF-&alpha, The compounds at a concentration of 25 &micro, M, except for shanzhiside methyl ester (6), inhibited secretion of IL-8 in the range from 29.1 to 50.0%. In conclusion, L. album might be a valuable source of bioactive compounds and may provide constituents to limit noninfectious inflammation associated with the aforementioned diseases.

Introduction The genus Ligustrum (Oleaceae) is distributed in Europe and Asia (south China and Korea), where it is used to prevent hypertension, sore throats, inflammation and diabetes. The main groups of compounds in extracts of Ligustrum vulgare are biologically active secoiridoids and phenylpropanoids. Objectives The aim of the study was primarily the development and validation of a HPTLC–photodensitometry method for separation and determination of secoiridoids (oleacein, oleuropein) and phenylpropanoids (echinacoside) in different extracts prepared from leaves of L. vulgare. A secondary issue was the quantitative screening of oleacein, oleuropein and echinacoside in extracts from leaves collected at different stages of plant growth (from May to September). Methods A HPTLC–photodensitometry method was developed and validated for quantification of oleuropein, oleacein and echinacoside in plant extracts (aqueous and ethanolic extract, decoction, infusion). Silica gel was used as the stationary phase and dichloromethane:methanol:formic acid:water (80:25:1.5:4, v/v/v/v) as the mobile phase. Results The HPTLC–photodensitometry method developed for quantification of oleacein, oleuropein and echinacoside was specific, accurate and precise. The presence of oleacein was detected in aqueous extracts, whereas oleuropein was present, in particular, in ethanolic extracts, decoctions and infusions. Echinacoside was detected in all the extracts prepared. The content of secoiridoids was variable from May to September, whereas the amount of echinacoside increased in this term. Conclusion The developed and validated HPTLC–photodensitometry method allowed performing fast screening of quantitative profiles of oleacein, oleuropein and echinacoside in preparations of privet leaves. Copyright © 2015 John Wiley & Sons, Ltd.

Background Chronic hyperalgesia and allodynia associated with progressive damage of peripheral neurons are the most prevalent complications of diabetes mellitus. Plants belonging to the family of Oleaceae were traditionally used in folk medicine for the management of diabetes. Hypothesis/Purpose The aim of this study was to investigate whether an aqueous extract from the leaves of Ligustrum vulgare (common privet) could be useful to target neuropathic pain in a rat streptozotocin (STZ) model of diabetes. Methods The chemical composition of the aqueous extract from privet leaf was characterized with the UHPLC-DAD-MS method and the analytical quantification of its constituents was performed with HPLC-DAD. Mechanical hyperalgesia and allodynia were evaluated with the Randall–Selitto and von Frey tests. Results Our investigation revealed the presence of secoiridoids: oleacein (23.48 ± 0.87 mg/g), oleocanthal (8.44 ± 0.08 mg/g), oleuropein (1.50 ± 0.01 mg/g), as well as phenylpropanoids: echinacoside (6.46 ± 0.07 mg/g), verbascoside (4.03 ± 0.04 mg/g) and p-coumaroyl glucarates in the dried aqueous extract of privet leaves. Behavioral data indicated that chronic intraperitoneal administration of the extract (50–200 mg/kg) for 21 days resulted in a decrease in diabetes-induced hyperalgesia and allodynia. Blood glucose levels remained unaltered, while body weight and water intake decreased significantly. Conclusion The aqueous privet leaf extract could serve useful in facilitating treatment of painful diabetic neuropathy. Additionally, the study showed that the antihyperalgesic activity of Ligustrum vulgare leaf extract is not likely related to its antihyperglycemic properties.

Thirteen new compounds including caffeoyl-glucaric and p-coumaroyl-altraric acid derivatives, one monoterpenoid glucoside, four secoiridoid glycosides, and three hydroxycinnamoyl phenylpropanoid glycosides esterified with an oleoside 11-methyl ester along with fifteen known compounds were isolated from flowers of Syringa vulgaris L. (Oleaceae). Their structures were elucidated by high-resolution spectroscopic methods. The tested compounds were able to decrease the production of reactive oxygen species. Moreover, oleoechinacoside (13), demethylhydroxyoleonuezhenide (14), demethyloleonuezhenide (15), syringaoleoacteoside (25) and oleoacteoside (26) at the concentration of 50μM, moderately suppressed the LPS-stimulated release of pro-inflammatory chemokine IL-8 and TNF-α from human neutrophils. Moreover, oleonuezhenide (12), oleoside 11-methyl ester (16) and oleoacteoside (26) at the concentration of 50μM were able to induce the surface expression of interleukin 10 receptor, which is suppressed by the incubation of monocyte/macrophage cells with LPS.

In traditional medicine, flowers and aerial parts of Lamium album are assigned by their anti-inflammatory, antiseptic, and mucolytic activities, and are used in chronic bronchitis and pharyngitis as well as skin, vaginal, and cervical inflammation.The aim of the present study was to compare effects of ethanolic extracts prepared from flowers and aerial parts of L. album on selected functions of human neutrophils, which are involved in an inflammatory response. In order to identify the compounds engaged in the anti-inflammatory activity of extracts, the bioassay-guided isolation of compounds was performed based on the inhibition of cytokine secretion by stimulated neutrophils.The extracts were phytochemically characterized with the HPLC-DAD-MSn method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase secretion by neutrophils was established spectrophotometrically. The levels of cytokine (interleukin 8, TNF-α) production after extract treatment was measured by ELISA.The most abundant constituents of extracts were phenylpropanoids, iridoids, flavonoids, and phenolic acids. Both extracts at concentrations of 25 and 100 µg/mL significantly inhibited reactive oxygen species production, and myeloperoxidase and interleukin 8 secretion. The phenylethanoid glycosides, such as lamiusides A, B, and C as well as 6″-O-β-D-glucopyranosylmartynoside, were isolated and identified. The cells treated with 6″-O-β-D-glucopyranosylmartynoside and lamiuside B produced 29.47 ± 7.11 % and 64.67 ± 5.25 % of interleukin 8, respectively, compared to non-treated control cells.Our results support the traditional use of L. album and indicate it as a potential source of natural anti-inflammatory constituents, such as phenylpropanoids.

We investigated in vitro the ability of a standardised olive leaf dry extract (Ph. Eur.) (OLE) as well as of its single components to circumvent the hydrogen peroxide-induced inhibition of the hypothiocyanite-producing activity of lactoperoxidase (LPO). The rate of hypothiocyanite (⁻OSCN) formation by LPO was quantified by spectrophotometric detection of the oxidation of 5-thio-2-nitrobenzoic acid (TNB). By using excess hydrogen peroxide, we forced the accumulation of inactive enzymatic intermediates which are unable to promote the two-electronic oxidation of thiocyanate. Both OLE and certain extract components showed a strong LPO-reactivating effect. Thereby an o-hydroxyphenolic moiety emerged to be essential for a good reactivity with the inactive LPO redox states. This basic moiety is found in the main OLE components oleuropein, oleacein, hydroxytyrosol, caffeic acid as well as in different other constituents including the OLE flavone luteolin. As LPO is a key player in the humoral immune response, these results propose a new mode of action regarding the well-known bacteriostatic and anti-inflammatory properties of the leaf extract of Olea europaea L.

Leaves of Ligustrum vulgare (common privet) have been used for treatment of oropharyngeal inflammations or as antirheumatic, diuretic, and hypotensive agents in folk medicine in southern Europe. Taking into account that neutrophils are involved in the inflammation, the aim of the study was to determine the effect of an aqueous extract prepared from leaves of Ligustrum vulgare on neutrophil functions. The extract was characterized by the HPLC-DAD-MSn method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase, metalloproteinase 9, and interleukin 8 production by neutrophils was measured by an enzyme-linked immunosorbent assay. Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules on neutrophils was analyzed with flow cytometry. The main compounds detected were flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. The inhibition of oxidative burst by the extract was comparable in both stimuli models (formyl-met-leu-phenylalanine: IC50 = 18.2 ± 4.0 µg/mL; phorbol 12-myristate 13-acetate: IC50 = 19.8 ± 3.0 µg/mL). The extract in the concentration range of 5–50 µg/mL inhibited neutrophil elastase release by 23.9–34.1 % and myeloperoxidase release by 24.2–37.4 %. The inhibitory effect on metalloproteinase 9 and interleukin 8 production was around 20 %. The extract in the highest concentration modulated the expression of L-selectin and β 2 integrin. Our results partly support the traditional use of common privet leaves as an anti-inflammatory agent.

In the present study we investigated the chemical composition of extracts prepared from aerial parts of Oenothera paradoxa Hudziok and Oenothera biennis L. and their antioxidative and anti-inflammatory activities. Ultra high pressure liquid chromatography (UHPLC)-DAD-MS/MS studies showed that both extracts contain a wide variety of polyphenols (39 identified constituents) among which macrocyclic ellagitannin turned out to be the main constituent. During the in vitro studies, using noncellular models, both extracts scavenged reactive oxygen species (ROS) in a concentration-dependent manner, and the lowest SC(50) values were obtained for O(2)(-) and H(2)O(2). Both extracts inhibited ROS production by stimulated human neutrophils. The stronger activity in the case of formyl-met-leu-phenylalanine stimulation suggests that both extracts may act through the receptor-dependent pathway. O. paradoxa extract and O. biennis extract exhibited anti-inflammatory activity by the inhibition of hyaluronidase and lipoxygenase in a concentration-dependent manner. The stronger activity of O.biennis extract toward lipoxygenase may be explained by its higher oenothein B content.

Background In patients with hypertension the haemorrhage into carotid atherosclerotic plaque increases risk of plaque destabilization and rupture. Our previous study showed that oleacein, a secoiridoid present in extra virgin olive oil, enhanced uptake of haemoglobin-haptoglobin complex and change macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2. Purpose The aim this study was to investigate a potential role of oleacein in attenuation of carotid plaque destabilisation ex vivo . Methods Samples of atherosclerotic plaque were harvested from 20 patients with hypertension /11 women and 9 men/, who underwent carotid endarterectomy after transient ischemic attacks. Matching pieces of each plaque were incubated with increased concentration of pure oleacein /range 0–20 µM/ for 24 h. HMGB1, MMP-9, MMP-9/NGAL, TF and IL-10, as well as HO-1 secretion from plaque was measured by enzyme–linked immunosorbent assay /ELISA/. Statistical significance was set at P 0.05 and P 0.001. Results Oleacein at the concentrations of 10 and 20 µM significantly ( P 0.001) decreased secretion of HMGB1 (up 90%), MMP-9 (up to 80%), MMP-9/NGAL complex (up to 80%) and TF (more than 90%) from the treated plaque, as compared to control. At the same time IL-10 and HO-1 release increased by more than 80% ( P 0.001). Conclusion Our results indicate that oleacein possess ability to attenuate the destabilization of carotid plaque and could be potentially useful in the reduction of ischemic stroke risk.

Phenolic compounds from virgin olive oil are known to play an antioxidant role in preventing biomolecule damage. One of the most abundant components of olive oil is an oleuropein derivative named oleacein (dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol; 3,4-DHPEA-EDA). The aim of the study was to establish the antioxidant activity of oleacein for an array of reactive oxygen (superoxide anion, O 2 - ; hydrogen peroxide, H2O2; hypochlorous acid, HOCl) and nitrogen species (nitric oxide, NO; peroxynitrite, ONOO−) formation, using in vitro non-cellular systems, as well as cellular screening systems (human neutrophil oxidative burst, monocytes’ nitric oxide production). We used a well-known antioxidant, oleuropein, as a reference compound. Oleacein proved to be stronger in the reduction of formyl-met-leu-phenylalanine (f-MLP) and phorbol–myristate–acetate (PMA)-induced oxidative bursts in neutrophils (IC50 = 1.8 μM, IC50 = 1.5 μM, respectively) and myeloperoxidase release (IC50 = 8.8 μM) than was oleuropein (IC50 = 2.4 μM, IC50 = 16.0 μM, IC50 = 30.7 μM, respectively). Both compounds were stronger scavengers of O 2 - than H2O2. Oleuropein did not scavenge HOCl as opposed to oleacein (IC50 = 1.5 μM). Both compounds, in a concentration range 1–10 μM, significantly decreased nitrogen species formation in cell-free systems and by lipopolysaccharide (LPS)-stimulated monocytes.

In this study, we analyzed ex vivo the effect of an aqueous extract of Oenothera paradoxa defatted seeds on the formation of neutrophil-derived oxidants. For defining active compounds, we also tested lypophilic extract constituents such as gallic acid, (+)-catechin, ellagic acid, and penta-O-galloyl-β-D-glucose and a hydrophilic fraction containing polymeric procyanidins. The anti-inflammatory potential of the extract and compounds was tested by determining the release from activated neutrophils of elastase, myeloperoxidase, interleukin-8 (IL-8), and leukotriene B4 (LTB4), which are considered relevant for the pathogenesis of cardiovascular diseases. The extract of O. paradoxa defatted seeds displays potent antioxidant effects against both 4β-phorbol-12β-myristate-α13-acetate- and formyl-met-leu-phenylalanine-induced reactive oxygen species production in neutrophils with IC50 values around 0.2 μg/mL. All types of polyphenolics present in the extract contributed to the extract antioxidant activity. According to their IC50 values, penta-O-galloyl-β-D-glucose was the more potent constituent of the extract. In cell-free assays, we demonstrated that this effect is partially due to the scavenging of O2- and H2O2 oxygen species. The extract and especially penta-O-galloyl-β-D-glucose significantly inhibit elastase, myeloperoxidase IL-8, and LTB4 release with an IC50 for penta-O-galloyl-β-D-glucose of 17±1, 15±1, 6.5±2.5, and around 20 μM, respectively. The inhibition of penta-O-galloyl-β-D-glucose on reactive oxygen species and especially on O2- production, myeloperoxidase, and chemoattractant release may reduce the interaction of polymorphonuclear leukocyte with the vascular endothelium and by that potentially diminish the risk of progression of atherosclerosis development.