Phenolic compound profiling and antioxidant potential of different types of Schisandra henryi in vitro cultures.

Schisandra henryi is an endemic species of medicinal potential known from traditional Chinese medicine. As part of this study, a complex biotechnological and phytochemical assessment was conducted on S. henryi with a focus on phenolic compounds and antioxidant profiling. The following in vitro cultures were tested: microshoot agar and callus, microshoot agitated, and suspension, along with the microshoot culture in PlantForm bioreactors. Qualitative profiling was performed by ultra-high-performance liquid chromatography with a photodiode array detector coupled with ion-trap mass spectrophotometry with electrospray ionization and then quantitative analysis by high-performance liquid chromatography with a diode array detector using standards. In the extracts, mainly the compounds from procyanidins were identified as well as phenolic acids (neochlorogenic acid, caffeic acid, protocatechuic acid) and catechin. The highest content of phenolic compounds was found for in vitro agar microshoot culture (max. total content 229.87 mg/100 g DW) and agitated culture (max. total content 22.82 mg/100 g DW). The max. TPC measured using the Folin-Ciocalteu assay was equal to 1240.51 mg GAE/100 g DW (agar microshoot culture). The extracts were evaluated for their antioxidant potential by the DPPH, FRAP, and chelate iron ion assays. The highest potential was indicated for agar microshoot culture (90% of inhibition and 59.31 nM/L TEAC, respectively). The research conducted on the polyphenol profiling and antioxidant potential of S. henryi in vitro culture extracts indicates the high therapeutic potential of this species. KEY POINTS: • Different types of S. henryi in vitro cultures were compared for the first time. • The S. henryi in vitro culture strong antioxidant potential was determined for the first time. • The polyphenol profiling of different types of S. henryi in vitro cultures was shown.

In Vitro and In Vivo Pharmacological Profiles of LENART01, a Dermorphin-Ranatensin Hybrid Peptide.

Diverse chemical and pharmacological strategies are currently being explored to minimize the unwanted side effects of currently used opioid analgesics while achieving effective pain relief. The use of multitarget ligands with activity at more than one receptor represents a promising therapeutic approach. We recently reported a bifunctional peptide-based hybrid LENART01 combining dermorphin and ranatensin pharmacophores, which displays activity to the mu-opioid receptor (MOR) and dopamine D2 receptor (D2R) in rat brains and spinal cords. In this study, we investigated the in vitro binding and functional activities to the human MOR and the in vivo pharmacology of LENART01 in mice after subcutaneous administration. In vitro binding assays showed LENART01 to bind and be selective to the human MOR over the other opioid receptor subtypes and delta, kappa and nociceptin receptors. In the [35S]GTPγS binding assay, LENART01 acted as a potent and full agonist to the human MOR. In mice, LENART01 produced dose-dependent antinociceptive effects in formalin-induced inflammatory pain, with increased potency than morphine. Antinociceptive effects were reversed by naloxone, indicating MOR activation in vivo. Behavioral studies also demonstrated LENART01's properties to induce less adverse effects without locomotor dysfunction and withdrawal syndrome compared to conventional opioid analgesics, such as morphine. LENART01 is the first peptide-based MOR-D2R ligand known to date and the first dual MOR-dopamine D2R ligand for which in vivo pharmacology is reported with antinociceptive efficacy and reduced opioid-related side effects. Our current findings may pave the way to new pain therapeutics with limited side effects in acute and chronic use.

In Vitro Antiviral Activity of Kalanchoe daigremontiana Extract against Human Herpesvirus Type 1.

Plant polyphenols possess diverse bioactivities, including antiviral activity against a broad spectrum of viruses. Here, we investigated the virucidal properties of an Kalanchoe daigremontiana extract using an in vitro model of human herpesvirus type 1 (HHV-1) infection. Chromatographic analysis indicated that the extract of Kalanchoe daigremontiana is rich in various compounds, among which are polyphenols with virucidal activity confirmed in the literature. We found that Kalanchoe daigremontiana extract shows an ability to prevent HHV-1 infection by direct inhibition of the virus attachment, penetration, and blocking of infection when used in pretreatment or post-entry treatment. Our results indicate that Kalanchoe daigremontiana extract may be a good candidate drug against HHV-1, both as a substance to prevent infection and to treat an already ongoing infection. Our findings illustrate that Kalanchoe daigremontiana could be a potential new candidate for clinical consideration in the treatment of HHV-1 infection alone or in combination with other therapeutics.

Gut Microbiota of Pigs Metabolizes Extracts of Filipendula ulmaria and Orthosiphon aristatus-Herbal Remedies Used in Urinary Tract Disorders.

Urinary tract infections influence the mortality rate in pigs and are linked to extensive antibiotic usage in the farm industry. Filipendula ulmaria (L.) Maxim. and Orthosiphon aristatus (Blume) Miq. are widespread medicinal plants traditionally used to treat urinary tract disorders. As their preparations are orally administered, the metabolism of their constituents by gut microbiota before absorption should be considered. Until now, no experiments had been performed to describe the biotransformation of tthose plants' extracts by animal gut microbiota. The study evaluates the influence of pig intestinal microbiota on the structure of active compounds in flowers of F. ulmaria and leaves of O. aristatus. The incubations of the extracts with piglet gut microbiota were performed in anaerobic conditions, and the samples of the batch culture were collected for 24 h. In F. ulmaria, the main metabolites were quercetin and kaempferol, which were products of the deglycosylation of flavonoids. After 24 h incubation of O. aristatus extract with the piglet gut microbiota, 2 main metabolites were observed. One, tentatively identified as 3-(3-dihydroxyphenyl)propionic acid, is likely the primary metabolite of the most abundant depsides and phenolic acids. The results confirm the formation of the compounds with anti-inflammatory and diuretic activity in the microbiota cultures, which might suggest F. ulmaria and O. aristatus for treating urinary tract disorders in piglets. Based on the similarities of human and pig gut microbiota, the pig model can help estimate the metabolic pathways of natural products in humans.

Current Knowledge on Interactions of Plant Materials Traditionally Used in Skin Diseases in Poland and Ukraine with Human Skin Microbiota.

Skin disorders of different etiology, such as dermatitis, atopic dermatitis, eczema, psoriasis, wounds, burns, and others, are widely spread in the population. In severe cases, they require the topical application of drugs, such as antibiotics, steroids, and calcineurin inhibitors. With milder symptoms, which do not require acute pharmacological interventions, medications, dietary supplements, and cosmetic products of plant material origin are gaining greater popularity among professionals and patients. They are applied in various pharmaceutical forms, such as raw infusions, tinctures, creams, and ointments. Although plant-based formulations have been used by humankind since ancient times, it is often unclear what the mechanisms of the observed beneficial effects are. Recent advances in the contribution of the skin microbiota in maintaining skin homeostasis can shed new light on understanding the activity of topically applied plant-based products. Although the influence of various plants on skin-related ailments are well documented in vivo and in vitro, little is known about the interaction with the network of the skin microbial ecosystem. The review aims to summarize the hitherto scientific data on plant-based topical preparations used in Poland and Ukraine and indicate future directions of the studies respecting recent developments in understanding the etiology of skin diseases. The current knowledge on investigations of interactions of plant materials/extracts with skin microbiome was reviewed for the first time.

Comparative Study of Chemical Stability of a PK20 Opioid-Neurotensin Hybrid Peptide and Its Analogue [Ile9]PK20-The Effect of Isomerism of a Single Amino Acid.

Chemical stability is one of the main problems during the discovery and development of potent drugs. When ignored, it may lead to unreliable biological and pharmacokinetics data, especially regarding the degradation of products' possible toxicity. Recently, two biologically active drug candidates were presented that combine both opioid and neurotensin pharmacophores in one entity, thus generating a hybrid compound. Importantly, these chimeras are structurally similar except for an amino acid change at position 9 of the peptide chain. In fact, isoleucine (C6H13NO2) was replaced with its isomer tert-leucine. These may further lead to various differences in hybrids' behavior under specific conditions (temperature, UV, oxidative, acid/base environment). Therefore, the purpose of the study is to assess and compare the chemical stability of two hybrid peptides that differ in nature by way of one amino acid (tert-leucine vs. isoleucine). The obtained results indicate that, opposite to biological activity, the substitution of tert-leucine into isoleucine did not substantially influence the compound's chemical stability. In fact, neither hydrolysis under alkaline and acidic conditions nor oxidative degradation resulted in spectacular differences between the two compounds-although the number of potential degradation products increased, particularly under acidic pH. However, such a modification significantly reduced the compound's half-life from 204.4 h (for PK20 exposed to 1M HCl) to 117.7 h for [Ile9]PK20.

Determination of Flavonoids in Selected Scleranthus Species and Their Anti-Collagenase and Antioxidant Potential.

A new 5,7-dihydroxy-3'-methoxy-4'-acetoxyflavone-8-C-ß-d-arabinopyranoside-2″-O-(4‴-acetoxy)-glucoside (6) and three known flavone C-glycosides-5,7,3',4'-tetrahydroxyflavone-6-C-xyloside-8-C-ß-d-glucoside (lucenin-1) (7), 5,7,3'-trihydroxyflavone-6-C-glucoside-8-C-ß-d-glucoside (vicenin-2) (8), and 5,7,4'-trihydroxy-3'-methoxyflavone-6-C-ß-d-glucopyranoside-8-C-α-arabinopyranoside (chrysoeriol-6-C-ß-d-glucopyranoside-8-C-α-arabinopyranoside) (9)-were isolated from aerial parts of Scleranthus perennis L. (Caryophyllaceae). Their structures were determined through the use of comprehensive spectroscopic and spectrometric methods, and a method for the quantification of the major constituents of S. perennis and S. annuus L. was developed. Furthermore, the anti-collagenase and antioxidant activities of all isolated compounds obtained from extracts and fractions from both Scleranthus species were evaluated. The highest percentage of collagenase inhibition (at 400 µg/mL) was distinguished for methanolic extracts (22.06%, 32.04%) and ethyl acetate fractions (16.59%, 14.40%) from S. annuus and S. perennis. Compounds 6-9 displayed moderate inhibitory activity, with IC50 values ranging from 39.59-73.86 µM.

Polyphenols and Maillard Reaction Products in Dried Prunus spinosa Fruits: Quality Aspects and Contribution to Anti-Inflammatory and Antioxidant Activity in Human Immune Cells Ex Vivo.

Dried Prunus spinosa fruits (sloes) are folk phytotherapeutics applied to treat chronic inflammatory disorders. However, their pharmacological potential, activity vectors, and drying-related changes in bioactive components remain unexplored. Therefore, the present research aimed to evaluate the anti-inflammatory and antioxidant effects of dried sloes in ex vivo models of human neutrophils and peripheral blood mononuclear cells (PMBCs) and establish their main active components. It was revealed that the fruit extracts significantly and dose-dependently inhibited the respiratory burst, downregulated the production of elastase (ELA-2) and TNF-α, and upregulated the IL-10 secretion by immune cells under pro-inflammatory and pro-oxidant stimulation. The slightly reduced IL-6 and IL-8 secretion was also observed. The structural identification of active compounds, including 45 phenolics and three Maillard reaction products (MRPs) which were formed during drying, was performed by an integrated approach combining LC-MS/MS, preparative HPLC isolation, and NMR studies. The cellular tests of four isolated model compounds (chlorogenic acid, quercetin, procyanidin B2, and 5-hydroxymethylfurfural), supported by statistical correlation studies, revealed a significant polyphenolic contribution and a slight impact of MRPs on the extracts' effects. Moreover, a substantial synergy was observed for phenolic acids, flavonoids, condensed proanthocyanidins, and MPRs. These results might support the phytotherapeutic use of dried P. spinosa fruits to relieve inflammation and establish the quality control procedure for the extracts prepared thereof.

The Effect of Standardised Leaf Extracts of Gaultheria procumbens on Multiple Oxidants, Inflammation-Related Enzymes, and Pro-Oxidant and Pro-Inflammatory Functions of Human Neutrophils.

The leaves of Gaultheria procumbens are polyphenol-rich traditional medicines used to treat inflammation-related diseases. The present study aimed to optimise the solvent for the effective recovery of active leaf components through simple direct extraction and verify the biological effects of the selected extract in a model of human neutrophils ex vivo. The extracts were comprehensively standardised, and forty-one individual polyphenols, representing salicylates, catechins, procyanidins, phenolic acids, and flavonoids, were identified by UHPLC-PDA-ESI-MS3. The chosen methanol-water (75:25, v/v) extract (ME) was obtained with the highest extraction yield and total phenolic levels (397.9 mg/g extract's dw), including 98.9 mg/g salicylates and 299.0 mg/g non-salicylate polyphenols. In biological tests, ME revealed a significant and dose-dependent ability to modulate pro-oxidant and pro-inflammatory functions of human neutrophils: it strongly reduced the ROS level and downregulated the release of pro-inflammatory cytokines and tissue remodelling enzymes, especially IL-1ß and elastase 2, in cells stimulated by fMLP, LPS, or fMLP + cytochalasin B. The extracts were also potent direct scavengers of in vivo relevant oxidants (O2•-, •OH, and H2O2) and inhibitors of pro-inflammatory enzymes (cyclooxygenase-2, hyaluronidase, and lipoxygenase). The statistically significant correlations between the tested variables revealed the synergic contribution of individual polyphenols to the observed effects and indicated them as useful active markers for the standardisation of the extract/plant material. Moreover, the safety of ME was confirmed in cytotoxicity tests. The obtained results might partially explain the ethnomedicinal application of G. procumbens leaves and support the usage of the standardised leaf extract in the adjuvant treatment of oxidative stress and inflammation-related chronic diseases.

Polyphenol-Enriched Extracts of Prunus spinosa Fruits: Anti-Inflammatory and Antioxidant Effects in Human Immune Cells Ex Vivo in Relation to Phytochemical Profile.

The fresh fruits of Prunus spinosa L., a wild plum species, are traditionally used for dietary purposes and medicinal applications in disorders related to inflammation and oxidative stress. This study aimed to investigate the phytochemical composition of the fruits in the function of fractionated extraction and evaluate the biological potential of the extracts as functional products in two models of human immune cells ex vivo. Fifty-seven phenolic components were identified in the extracts by UHPLC-PDA-ESI-MS3, including twenty-eight new for the analysed fruits. Fractionation enabled the enrichment of polyphenols in the extracts up to 126.5 mg gallic acid equivalents/g dw total contents, 91.3 mg/g phenolic acids (caffeoyl-, coumaroyl-, and feruloylquinic acids), 41.1 mg/g flavonoids (mostly quercetin mono-, di- and triglycosides), 44.5 mg/g condensed proanthocyanidins, and 9.2 mg/g anthocyanins (cyanidin and peonidin glycosides). The hydroalcoholic extract and phenolic-enriched fractions of the fruits revealed significant ability to modulate pro-oxidant, pro-inflammatory, and anti-inflammatory functions of human neutrophils and peripheral blood mononuclear cells (PBMCs): they strongly downregulated the release of reactive oxygen species, TNF-α, and neutrophils elastase, upregulated the secretion of IL-10, and slightly inhibited the production of IL-8 and IL-6 in the cells stimulated by fMLP, fMLP+cytochalasin B, and LPS, depending on the test. Correlation studies and experiments on the pure compounds indicated a significant contribution of polyphenols to these effects. Moreover, cellular safety was confirmed for the extracts by flow cytometry in a wide range of concentrations. The results support the traditional use of fresh blackthorn fruits in inflammatory disorders and indicate extracts that are most promising for functional applications.

Bioactive Potential: A Pharmacognostic Definition through the Screening of Four Hypericum Species from the Canary Islands.

In this work, we propose a general methodology to assess the bioactive potential (BP) of extracts in the quest of vegetable-based drugs. To exemplify the method, we studied the anticancer potential (AP) of four endemic species of genus Hypericum (Hypericum canariense L, Hypericum glandulosum Aiton, Hypericum grandifolium Choisy and Hypericum reflexum L.f) from the Canary Islands. Microextracts were obtained from the aerial parts of these species and were tested against six human tumor cell lines, A549 (non-small-cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small-cell lung), T-47D (breast) and WiDr (colon). The methanol-water microextracts were evaluated further for cell migration, autophagy and cell death. The most promising bioactive polar microextracts were analyzed by UHPLC-DAD-MS. The extraction yield, the bioactivity evaluation and the chemical profiling by LC-MS suggested that H. grandifolium was the species with the highest AP. Label-free live-cell imaging studies on HeLa cells exposed to the methanol-water microextract of H. grandifolium enabled observing cell death and several apoptotic hallmarks. Overall, this study allows us to select Hypericum grandifolium Choisy as a source of new chemical entities with a potential interest for cancer treatment.

Comparative studies of urolithins and their phase II metabolites on macrophage and neutrophil functions.

PURPOSE: Ellagitannins are high molecular weight polyphenols present in high quantities in various food products. They are metabolized by human and animal gut microbiota to postbiotic metabolites-urolithins, bioavailable molecules of a low molecular weight. Following absorption in the gut, urolithins rapidly undergo phase II metabolism. Thus, to fully evaluate the mechanisms of their biological activity, the in vitro studies should be conducted for their phase II conjugates, mainly glucuronides. The aim of the study was to comparatively determine the influence of urolithin A, iso-urolithin A, and urolithin B together with their respective glucuronides on processes associated with the inflammatory response. METHODS: The urolithins obtained by chemical synthesis or isolation from microbiota cultures were tested with their respective glucuronides isolated from human urine towards modulation of inflammatory response in THP-1-derived macrophages, RAW 264.7 macrophages, PBMCs-derived macrophages, and primary neutrophils. RESULTS: Urolithin A was confirmed to be the most active metabolite in terms of LPS-induced inflammatory response inhibition (TNF-α attenuation, IL-10 induction). The observed strong induction of ERK1/2 phosphorylation has been postulated as the mechanism of its action. None of the tested glucuronide conjugates was active in terms of pro-inflammatory TNF-α inhibition and anti-inflammatory IL-10 and TGF-ß1 induction. CONCLUSION: Comparative studies of the most abundant urolithins and their phase II conjugates conducted on human and murine immune cells unambiguously confirmed urolithin A to be the most active metabolite in terms of inhibition of the inflammatory response. Phase II metabolism was shown to result in the loss of urolithins' pharmacological properties.

Screening for the Active Anti-Inflammatory and Antioxidant Polyphenols of Gaultheria procumbens and Their Application for Standardisation: From Identification through Cellular Studies to Quantitative Determination.

Aerial parts, leaves, and stems of Gaultheria procumbens are polyphenol-rich herbal medicines with anti-inflammatory and antioxidant effects. The present study focused on identifying active markers of the G. procumbens extracts in an integrated approach combining phytochemical and biological capacity tests. The target compounds, representing all classes of Gaultheria polyphenols, were pre-selected by LC-ESI-PDA-MS/MS. For unambiguous identification, the key analytes, including a rare procyanidin trimer (cinnamtannin B-1), miquelianin potassium salt, and two new natural products: quercetin and kaempferol 3-O-ß-d-xylopyranosyl-(1→2)-ß-d-glucuronopyranosides, were isolated by preparative HPLC and investigated by spectroscopy (HR-ESI-MS, UV-vis, CD, 1D- and 2D-NMR), thiolysis, flame photometry, optical rotation experiments, and absolute configuration studies. The significant contribution of the pre-selected compounds to the biological effects of the extracts was confirmed in vitro: the analytes significantly and in a dose-dependent manner down-regulated the pro-oxidant and pro-inflammatory functions of human neutrophils ex vivo (inhibited the release of reactive oxygen species, IL-1ß, TNF-α, and neutrophils elastase, ELA-2), inhibited two key pro-inflammatory enzymes (cyclooxygenase, COX-2, and hyaluronidase), and most of them, except gaultherin, exerted potent direct antioxidant activity (ferric reducing antioxidant power and superoxide anion scavenging capacity). Moreover, cellular safety was confirmed for all compounds by flow cytometry. Eventually, as these mechanisms have been connected to the health benefits of G. procumbens, 11 polyphenols were accepted as active markers, and a simple, accurate, reproducible, and fully validated RP-HPLC-PDA method for standardisation of the target extracts was proposed.

New Flavone C-Glycosides from Scleranthus perennis and Their Anti-Collagenase Activity.

Three new flavone glycosides, one known flavone glycoside, and the phenolic derivative apiopaenonside were isolated and identified from the ethyl acetate fraction of the aerial parts of Scleranthus perennis. The planar structures were elucidated through extensive analysis of UV-Vis, IR, and 1H NMR and 13C NMR spectral data, including the 2D techniques COSY, HSQC, and HMBC, as well as ESI mass spectrometry. The isolated compounds were established as 5,7,3'-trihydroxy-4'-acetoxyflavone-8-C-ß-d-xylopyranoside-2''-O-glucoside (1), 5,7,3'-trihydroxy-4'-methoxyflavone-8-C-ß-d-xylopyranoside-2''-O-glucoside (2), 5,7-dihydroxy-3'-methoxy-4'-acetoxyflavone-8-C-ß-d-xylopyranoside-2''-O-glucoside (3), 5,7-dihydroxy-3'-methoxy-4'-acetoxyflavone-8-C-ß-d-xylopyranoside-2''-O-(4'''-acetoxy)-glucoside (4), and apiopaenonside (5). Moreover, all isolated compounds were evaluated for anti-collagenase activity. All compounds exhibited moderate inhibitory activity with IC50 values ranging from 36.06 to 70.24 µM.

Comprehensive Insight into Chemical Stability of Important Antidiabetic Drug Vildagliptin Using Chromatography (LC-UV and UHPLC-DAD-MS) and Spectroscopy (Mid-IR and NIR with PCA).

During forced degradation, the intrinsic stability of active pharmaceutical ingredients (APIs) could be determined and possible impurities that would occur during the shelf life of the drug substance or the drug product could be estimated. Vildagliptin belongs to relatively new oral antidiabetic drugs named gliptins, inhibiting dipeptidyl peptidase 4 (DPP-4) and prolonging the activities of the endogenous incretin hormones. At the same time, some gliptins were shown as prone to degradation under specific pH and temperature conditions, as well as in the presence of some reactive excipients. Thus, forced degradation of vildagliptin was performed at high temperature in extreme pH and oxidative conditions. Then, selective LC-UV was used for quantitative determination of non-degraded vildagliptin in the presence of its degradation products and for degradation kinetics. Finally, identification of degradation products of vildagliptin was performed using an UHPLC-DAD-MS with positive ESI. Stability of vildagliptin was also examined in the presence of pharmaceutical excipients, using mid-IR and NIR with principal component analysis (PCA). At 70 °C almost complete disintegration of vildagliptin occurred in acidic, basic, and oxidative media. What is more, high degradation of vildagliptin following the pseudo first-order kinetics was observed at room temperature with calculated k values 4.76 × 10-4 s-1, 3.11 × 10-4 s-1, and 1.73 × 10-4 s-1 for oxidative, basic and acidic conditions, respectively. Next, new degradation products of vildagliptin were detected using UHPLC-DAD-MS and their molecular structures were proposed. Three degradants were formed under basic and acidic conditions, and were identified as [(3-hydroxytricyclo- [3.3.1.13,7]decan-1-yl)amino]acetic acid, 1-{[(3-hydroxytricyclo[3.3.1.13,7]decan-1-yl)amino]acetyl}-pyrrolidine-2-carboxylic acid and its O-methyl ester. The fourth degradant was formed in basic, acidic, and oxidative conditions, and was identified as 1-{[(3-hydroxytricyclo[3.3.1.13,7]-decan-1-yl)amino]acetyl}pyrrolidine-2-carboxamide. When stability of vildagliptin was examined in the presence of four excipients under high temperature and humidity, a visible impact of lactose, mannitol, magnesium stearate, and polyvinylpirrolidone was observed, affecting-NH- and CO groups of the drug. The obtained results (kinetic parameters, interactions with excipients) may serve pharmaceutical industry to prevent chemical changes in final pharmaceutical products containing vildagliptin. Other results (e.g., identification of new degradation products) may serve as a starting point for qualifying new degradants of vildagliptin as it is related to substances in pharmacopoeias.

Lythrum salicaria Ellagitannins Stimulate IPEC-J2 Cells Monolayer Formation and Inhibit Enteropathogenic Escherichia coli Growth and Adhesion.

Lythrum salicaria herb (LSH) was applied in diarrhea therapy since ancient times. Despite empirically referenced therapeutic effects, the bioactivity mechanisms and chemical constituents responsible for pharmacological activity remain not fully resolved. Taking into consideration the historical use of LSH in treatment of diarrhea in humans and farm animals, the aim of the study was to examine in vitro the influence of LSH and its C-glycosylic ellagitannins on processes associated with maintaining intestinal epithelium integrity and enteropathogenic Escherichia coli (EPEC) growth and adhesion. LSH was not only inhibiting EPEC growth in a concentration dependent manner but also its adhesion to IPEC-J2 intestinal epithelial cell monolayers. Inhibitory activity toward EPEC growth was additionally confirmed ex vivo in distal colon samples of postweaning piglets. LSH and its dominating C-glycosylic ellagitannins, castalagin (1), vescalagin (2), and salicarinins A (3) and B (4) were stimulating IPEC-J2 monolayer formation by enhancing claudin 4 production. Parallelly tested gut microbiota metabolites of LSH ellagitannins, urolithin C (5), urolithin A (6), and its glucuronides (7) were inactive. The activities of LSH and the isolated ellagitannins support its purported antidiarrheal properties and indicate potential mechanisms responsible for its beneficial influence on the intestinal epithelium.

Phenolic Composition of the Leaves of Pyrola rotundifolia L. and Their Antioxidant and Cytotoxic Activity.

The leaves of Pyrola rotundifolia L. were extracted in the mixed solvent of methanol/acetone/water (2:2:1, v/v/v) and investigated for their phytochemical analysis and biological activity. Total phenolic and flavonoid contents were determined spectrophotometrically. A high content of phenols (208.35 mg GAE/g of dry extract), flavonoids (38.90 mg QE/g of dry extract) and gallotannins (722.91 GAE/g of dry extract) was obtained. Ultra-high performance liquid chromatography diode array detector tandem mass spectrometry (UHPLC-DAD-MS) allowed for the detection of 23 major peaks at 254 nm. The extract was analyzed for its antioxidant capacity using 2,2-diphenyl-1-picryl-hydrazyl (DPPH•) and 2,2'-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS•+) radical scavenging, metal chelating power and ß-carotene-linoleic acid bleaching assays. The examined extract showed moderate radical scavenging and chelating activity, and good inhibiting ability of linoleic acid oxidation (EC50 = 0.05 mg/mL) in comparison to standards. The cytotoxic effect in increasing concentration on five types of leukemic cell lines was also investigated using trypan blue vital staining. It was found that the analyzed extract induced the apoptosis of all the tested cell lines. Our findings suggest that the leaves of P. rotundifolia are a source of valuable compounds providing protection against oxidative damage, hence their use in traditional medicine is justified.

Immunomodulatory Activity and Phytochemical Profile of Infusions from Cleavers Herb.

Extracts from aerial parts of G. aparine (cleavers) constitute a herbal remedy with monography in British Herbal Pharmacopeia. On the European market, there are several drugs and food supplements consisting of Galium extracts. In folk medicine, cleavers was used topically in Europe, Asia, and the Americas to treat skin diseases. In several remedies, cleavers is also listed as an immunomodulatory active herb influencing the defense response of the human body. The aim of this study was to investigate the immunostimulatory activity and antioxidant potential in vitro of a raw infusion of cleavers and bioactive fractions. The functional activity of lymphocytes in the reaction of the lymphocyte blast transformation (RLBT) method was used for immunomodulatory activity assays and direct scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydrogen peroxide (H2O2) was chosen for the examination of antioxidant activity. It was shown that both the raw extract and fractions show significant immunostimulatory and scavenging activities. The obtained data partially justify the traditional use of cleavers as topical remedy for skin infections and for wounds.

Phytochemical and Biological Activity Studies on Nasturtium officinale (Watercress) Microshoot Cultures Grown in RITA® Temporary Immersion Systems.

The main compounds in both extracts were gluconasturtiin, 4-methoxyglucobrassicin and rutoside, the amounts of which were, respectively, determined as 182.93, 58.86 and 23.24 mg/100 g dry weight (DW) in biomass extracts and 640.94, 23.47 and 7.20 mg/100 g DW in plant herb extracts. The antioxidant potential of all the studied extracts evaluated using CUPRAC (CUPric Reducing Antioxidant Activity), FRAP (Ferric Reducing Ability of Plasma), and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays was comparable. The anti-inflammatory activity of the extracts was tested based on the inhibition of 15-lipoxygenase, cyclooxygenase-1, cyclooxygenase-2 (COX-2), and phospholipase A2. The results demonstrate significantly higher inhibition of COX-2 for in vitro cultured biomass compared with the herb extracts (75.4 and 41.1%, respectively). Moreover, all the studied extracts showed almost similar antibacterial and antifungal potential. Based on these findings, and due to the fact that the growth of in vitro microshoots is independent of environmental conditions and unaffected by environmental pollution, we propose that biomass that can be rapidly grown in RITA® bioreactors can serve as an alternative source of bioactive compounds with valuable biological properties.

Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via Suppression of IL-8 and TNF-alpha Production and p38 and ERK 1/2 MAP Kinases.

During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 µM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the ß2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.