A double-blind randomized clinical trial of inflammatory cytokine and pepsin levels in the saliva of patients with voice prostheses.

BACKGROUND: Tracheoesophageal speech is one of the most effective method used for voice rehabilitation after laryngectomy. The main limitation is the need for periodic voice prothesis (VP) replacements. The process of developing VP usage complications is still unexplored. The aim of this study was to assess the level of cytokines (IL-1ß, IL-6, IL-8, IL-10, TNFα) and pepsin in saliva as potential factors reducing VP longevity. METHODS: Prospective double-blind randomized clinical trial was conducted (NCT04268459). Patients were randomly divided into two groups depending on VP replacement regimen (regular-every 3 months, or irregular-when complications occur). Levels of IL-1ß, IL-6, IL-8, IL-10, TNFα, and pepsin in saliva samples (fasting and after eating) of laryngectomized patients were measured using ELISA tests. RESULTS: Fifty-two patients (26 in both groups) with control group (7 patients) participated in the study. The level of IL-1ß, IL-6, IL-8, IL-10, TNFα, and pepsin did not differ according to regularity of VP replacements (p = 0.301-0.801). IL-6 levels were significantly higher when VP complications occurs (p = 0.012). CONCLUSIONS: The saliva components were not significantly different depending on the frequency of VP replacements. IL-6 plays an important role in the development of VP use complications.

Methodological Principles of Nasal Food Challenge.

Thanks to their valuable assessment possibilities (subjective complaints and changes in nasal patency during the examination), nasal provocation tests may serve as an alternative tool for oral food challenges in the future. However, this test requires successive attempts to regulate its methodology in order to develop a standardized lyophilisate form and determine the threshold dose for a positive result. The study objective was to present the methodological foundation for nasal food allergen provocation tests induced by freeze-dried powdered chicken egg whites. A control group of 25 individuals with no history of allergy to chicken eggs or any other allergy was included in the study. Optical rhinometry and visual analog scales were used to assess the response of nasal mucosa to local allergen challenges. Minor variations in nasal flows, as measured by optical rhinometry, were observed in the provocation tests. The mean optical density measurements (as measured regardless of the allergen dose used) varied from positive to negative values and vice versa, e.g., amounting to 0.018 OD (standard deviation 0.095) at 15 min and -0.011 OD (standard deviation 0.090) at 30 min. No significant differences were observed concerning the perceived nasal discomfort using the visual analog scale. Due to the absence of nasal mucosal reactivity, nasal challenge is an excellent methodological tool for implementing food allergen tests.

Role of Plant-Derived Compounds in the Molecular Pathways Related to Inflammation.

Inflammation is the primary response to infection and injury. Its beneficial effect is an immediate resolution of the pathophysiological event. However, sustained production of inflammatory mediators such as reactive oxygen species and cytokines may cause alterations in DNA integrity and lead to malignant cell transformation and cancer. More attention has recently been paid to pyroptosis, which is an inflammatory necrosis that activates inflammasomes and the secretion of cytokines. Taking into consideration that phenolic compounds are widely available in diet and medicinal plants, their role in the prevention and support of the treatment of chronic diseases is apparent. Recently, much attention has been paid to explaining the significance of isolated compounds in the molecular pathways related to inflammation. Therefore, this review aimed to screen reports concerning the molecular mode of action assigned to phenolic compounds. The most representative compounds from the classes of flavonoids, tannins, phenolic acids, and phenolic glycosides were selected for this review. Our attention was focused mainly on nuclear factor-κB (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), and mitogen-activated protein kinase (MAPK) signaling pathways. Literature searching was performed using Scopus, PubMed, and Medline databases. In conclusion, based on the available literature, phenolic compounds regulate NF-κB, Nrf2, and MAPK signaling, which supports their potential role in chronic inflammatory disorders, including osteoarthritis, neurodegenerative diseases, cardiovascular, and pulmonary disorders.

Influence of pyrrolizidine alkaloids depletion upon the biological activity of Symphytum officinale L. extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: In this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. MATERIALS AND METHODS: 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-1ß, IL-8, and TNF-α were determined (ELISA assay). RESULTS: 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-1ß, IL-8, and TNF-α) in LPS-stimulated neutrophils. CONCLUSIONS: In light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

Exploring the Artemisia Genus: An Insight into the Phytochemical and Multi-Biological Potential of A. campestris subsp. lednicensis (Spreng.) Greuter & Raab-Straube.

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.

Fruits of Hippophaë rhamnoides in human leukocytes and Caco-2 cell monolayer models-A question about their preventive role in lipopolysaccharide leakage and cytokine secretion in endotoxemia.

Preparations from Hippophaë rhamnoides L. (sea buckthorn) have been traditionally used in the treatment of skin and digestive disorders, such as gastritis, gastric and duodenal ulcers, uterine erosions, as well as oral, rectal, and vaginal mucositis, in particular in the Himalayan and Eurasian regions. An influence of an aqueous extract from the fruits of H. rhamnoides (HR) on leakage of lipopolysaccharide (LPS) from Escherichia coli through gut epithelium developed from the human colorectal adenocarcinoma (Caco-2) monolayer in vitro and glucose transporter 2 (GLUT2) translocation were the principal objectives of the study. Additionally, the effect of HR on the production of pro- and anti-inflammatory cytokines (interleukins: IL-8, IL-1ß, IL-10, IL-6; tumor necrosis factor: TNF-α) by the Caco-2 cell line, human neutrophils (PMN), and peripheral blood mononuclear cells (PBMC) was evaluated. The concentration of LPS on the apical and basolateral sides of the Caco-2 monolayer was evaluated with a Limulus Amebocyte Lysate (LAL) assay. GLUT2 translocation was evaluated using an immunostaining assay, whereas secretion of cytokines by cell cultures was established with an enzyme-linked immunosorbent (ELISA) assay. HR (500 µg/ml) significantly inhibited LPS leakage through epithelial monolayer in vitro in comparison with non-treated control. The treatment of Caco-2 cells with HR (50-100 µg/ml) showed GLUT2 expression similar to the non-treated control. HR decreased the secretion of most pro-inflammatory cytokines in all tested models. HR might prevent low-grade chronic inflammation caused by metabolic endotoxemia through the prevention of the absorption of LPS and decrease of chemotactic factors released by immune and epithelial cells, which support its use in metabolic disorders in traditional medicine.

Potentially Bio-Accessible Metabolites from an Extract of Cornus mas Fruit after Gastrointestinal Digestion In Vitro and Gut Microbiota Ex Vivo Treatment.

Targeting pancreatic lipase and α-amylase by digestion-derived fractions of ethanolic-aqueous (60%, v/v) extract from Cornus mas fruit (CM) in relation to the control and prevention of metabolic disorders, including diabetes, was the first purpose of the present study. Taking into consideration the significance of bio-accessibility of compounds, we attempted to identify metabolites of CM after gastrointestinal digestion in vitro, as well as their kinetic changes upon gut microbiota treatment. The digestion of extract was simulated with digestive enzymes in vitro and human gut microbiota ex vivo (1 h, 3 h, 6 h, 24 h), followed by chromatographic analysis using the UHPLC-DAD-MSn method. The effect of fractions from gastrointestinal digestion in vitro on the activity of pancreatic lipase and α-amylase was studied with fluorescence-based assays. The gastric and intestinal fractions obtained after in vitro digestion of CM inhibited pancreatic lipase and α-amylase. Loganic acid as the main constituent of the extract was digested in the experimental conditions in contrast to cornuside. It was found in most analytes such as salivary, gastric, intestinal, and even colon (fecal slurry, FS) fractions. In all fractions, kaempferol hexoside and reduced forms of kaempferol, such as aromadendrin, and benzoic acid were assigned. The signals of tannins were detected in all fractions. Cornusiin A was tentatively assigned in the gastric fraction. The metabolites originating from kinetic analytes have been classified mainly as phenolic acids, hydrolyzable tannins, and flavonoids. Phenolic acids (protocatechuic acid, gallic acid), tannins (digalloylglucose, tri-O-galloyl-ß-D-glucose), and flavonoids (aromadendrin, dihydroquercetin) were detected in the late phases of digestion in fecal slurry suspension. Cornuside was found in FS analyte after 3 h incubation. It was not detected in the samples after 6 and 24 h incubation with FS. In conclusion, cornuside, aromadendrin, and phenolic acids may be potentially bio-accessible compounds of CM. The presence of plants' secondary metabolites in the intestinal fractions allows us to indicate them as responsible for decreasing glucose and lipid absorption.

Sambucus nigra L. leaves inhibit TNF-α secretion by LPS-stimulated human neutrophils and strongly scavenge reactive oxygen species.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: 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. MATERIALS AND METHODS: The effect of extracts both on the secretion of cytokines (TNF-α, IL-1ß, 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-MSn and HPLC-DAD method, respectively. RESULTS: Elderberry 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 × mL-1 reduce the secretion of TNF-α by approximately 40% and 10%, respectively. ROS secretion was decreased by around 50% for all extracts at concentration of 5 µg × mL-1. All the extracts were able to inhibit the activity of lipoxygenase. The ethanolic extracts were characterized by a higher content of phenolic compounds and a higher antioxidant activity, especially against nitric oxide, compared to the aqueous extracts. CONCLUSIONS: Our 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.

Cornus mas and Cornus officinalis-A Comparison of Antioxidant and Immunomodulatory Activities of Standardized Fruit Extracts in Human Neutrophils and Caco-2 Models.

Fruits of Cornus mas and Cornus officinalis are representative plant materials traditionally used in Europe and Asia, respectively, in the treatment of diabetes and diabetes-related complications, which are often mediated by pathogenic inflammatory agents. Additionally, due to the fact of mutual infiltration of Asian and European medicines, the differentiation as well as standardization of traditional prescriptions seem to be crucial for ensuring the quality of traditional products. The objective of this study was a comparison of biological activity of extracts from fruits of C. mas and C. officinalis by an assessment of their effect on reactive oxygen species (ROS) generation in human neutrophils as well as cytokines secretion both in neutrophils (tumor necrosis factor α, TNF- α; interleukin 8, IL-8; interleukin 1ß, IL-1ß) and in human colon adenocarcinoma cell line Caco-2 (IL-8). To evaluate the phytochemical differences between the studied extracts as well as to provide a method for standardization procedures, a quantitative analysis of iridoids, such as loganin, sweroside, and loganic acid, found in extracts of Cornus fruits was performed with HPLC-DAD. All standardized extracts significantly inhibited ROS production, whereas the aqueous-alcoholic extracts were particularly active inhibitors of IL-8 secretion by neutrophils. The aqueous-methanolic extract of C. officinalis fruit, decreased IL-8 secretion by neutrophils to 54.64 ± 7.67%, 49.68 ± 6.55%, 50.29 ± 5.87% at concentrations of 5, 50, and 100 µg/mL, respectively, compared to LPS-stimulated control (100%). The aqueous extract of C. officinalis fruit significantly inhibited TNF-α release by neutrophils at concentrations of 50 and 100 µg/mL. On the other hand, the aqueous-ethanolic extract of C. mas fruit showed the propensity to increase TNF-α and IL-1ß secretion. The modulatory activity of the Cornus extracts was noted in the case of secretion of IL-8 in Caco-2 cells. The effect was comparable with dexamethasone. The content of loganin in aqueous and aqueous-methanolic extract of C. officinalis fruit was higher than in the aqueous-ethanolic extract of C. mas fruit, which was characterized by a significant quantity of loganic acid. In conclusion, the immunomodulatory effect observed in vitro may partially confirm the traditional use of Cornus fruits through alleviation of the development of diabetes-derived inflammatory complications. Loganin and loganic acid are significant markers for standardization of C. mas and C. officinalis fruit extracts, respectively.

Apiaceae Essential Oils: Boosters of Terbinafine Activity against Dermatophytes and Potent Anti-Inflammatory Effectors.

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-1ß, 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-1ß 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.

Honokiol and Magnolol: Insights into Their Antidermatophytic Effects.

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-1ß, 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-1ß, IL-8 and TNF-α released from lipopolysaccharide (LPS)-stimulated human neutrophils, while honokiol only decreased IL-1ß secretion, compared to the untreated control. Overall, honokiol and magnolol acted as fungicidal agents against dermatophytes, with impairment of ergosterol biosynthesis.

Screening of Antioxidative Properties and Inhibition of Inflammation-Linked Enzymes by Aqueous and Ethanolic Extracts of Plants Traditionally Used in Wound Healing in Poland.

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.

Pancreatic lipase and α-amylase inhibitory activity of extracts from selected plant materials after gastrointestinal digestion in vitro.

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 Hippophaë 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%.

Gut microbiota metabolism and the permeability of natural products contained in infusions from herb of European goldenrod Solidago virgaurea L.

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.

Inhibition of Neutrophil Functions and Antibacterial Effects of Tarragon (Artemisia dracunculus L.) Infusion-Phytochemical Characterization.

The aim of the study was to characterize phytochemicals in an infusion of the aerial parts of tarragon (Artemisia dracunculus L.) using ultra-high-performance liquid chromatography diode array detector electrospray ionisation tandem mass spectrometry UHPLC-DAD-ESI-MS/MS method, as well as an evaluation of its effects on mediators of the inflammation in an in vitro model of human neutrophils, and antimicrobial activity on selected pathogens. Flavonoids and caffeoylquinic acids were the main phenolic components of the extract of tarragon's aerial parts. The infusion was able to inhibit reactive oxygen species (ROS), interleukin 8 (IL-8), and tumour necrosis factor α (TNF-α) production. The antimicrobial assay was performed with the use of nine strains of bacteria, both Gram-negative and Gram-positive. Three human pathogens, Staphylococcus aureus ATCC6538, Staphylococcus epidermidis ATCC14990, and Staphylococcus aureus MRSA (methicyllin-resistant Staphylococcus aureus) ATCC43300, proved to be the most sensitive to tarragon infusion. Our study demonstrated the antiinflammatory and antimicrobial properties of tarragon (Artemisia dracunculus L.), meaning the common spice may be a prospective source of health-promoting constituents.

Symphytum officinale L.: Liquid-liquid chromatography isolation of caffeic acid oligomers and evaluation of their influence on pro-inflammatory cytokine release in LPS-stimulated neutrophils.

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)-1ß, 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-1ß 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.

Lamalbid, Chlorogenic Acid, and Verbascoside as Tools for Standardization of Lamium album Flowers-Development and Validation of HPLC-DAD Method.

Preparations from the flowers or herb of the white dead nettle (Lamium album L.) are recommended for the treatment of upper respiratory tract disorders or as a topical medication for mild inflammation of the throat, mouth, and skin. Taking into consideration the significance of L. album in traditional medicines across Europe, as well as the lack of studies describing the quantities of their most abundant constituents, we aimed to design a high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) method for potential standardization procedures of extracts from flowers of L. album. The HPLC-DAD method was developed and validated for quantification of iridoids (lamalbid), phenolic acids/depsides (chlorogenic acid), phenylpropanoids (verbascoside), and flavonoids (rutin; quercetin malonylhexoside; tiliroside) in aqueous and ethanolic-aqueous extracts of Lamii albi flos. The method was specific, accurate, and precise. Lamalbid was the most abundant compound both in aqueous (39.09 ± 1.02 m/g dry weight) and ethanolic-aqueous (26.66 ± 0.64 m/g dry weight) extracts. The quantities of selected compounds, except for chlorogenic acid and tiliroside, were higher in the aqueous extract than in the ethanolic-aqueous one. In conclusion, the method developed allowed for quantitation of compounds from different classes. In particular, chlorogenic acid and verbascoside have been proposed as reference compounds for routine quantitative control of Lamii albi flos.

Inhibition of Digestive Enzymes and Antioxidant Activity of Extracts from Fruits of Cornus alba, Cornus sanguinea subsp. hungarica and Cornus florida-A Comparative Study.

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 α-amylase, pancreatic lipase, and α-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 α-amylase was the most strongly inhibited by the fruit extract of CA (IC50 = 115.20 ± 14.31 µg/mL) and the activity of α-glucosidase by the fruit of CF (IC50 = 38.87 ± 2.65 µg/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.

Inula helenium and Grindelia squarrosa as a source of compounds with anti-inflammatory activity in human neutrophils and cultured human respiratory epithelium.

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-1ß 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-1ß 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 ß2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1ß 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.

In vitro α-amylase and pancreatic lipase inhibitory activity of Cornus mas L. and Cornus alba L. fruit extracts.

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.