Evaluation of the antioxidant activity of several naturally occurring coumarins and their synthesized analogues by "ferric reducing antioxidant power" assay.

Coumarins have attracted intense interest in recent years because of their diverse pharmacological properties. According to our continuing investigations of biological effects of several coumarins, the structure-antioxidant activity relationships (SARs) of six naturally occurring coumarins and their 16 synthesized analogues were established. For this purpose, the very reliable colorimetric assay (ferric reducing antioxidant power) modified to be used in 96-well microplates was used. This approach, which determines the reducing capacity of tested compounds directly, has previously been used for the determination of SARs of flavonoids, but has not been used for SAR determination of coumarins. It is known that the biological properties and consequently, therapeutic application of simple coumarins depends upon the pattern of substitution. It was established that 7,8-dihydroxy- and 6,7-dihydroxy-4-methylcoumarins have shown excellent ferric-reducing properties.

In vitro platelet antiaggregatory properties of 4-methylcoumarins.

Platelets play a crucial role in physiological haemostasis. However, in coronary arteries damaged by atherosclerosis, enhanced platelet aggregation, with subsequent thrombus formation, is a precipitating factor in acute myocardial infarction. Current therapeutic approaches are able to reduce approximately one quarter of cardiovascular events, but they are associated with an increased risk of bleeding and in some resistant patients are not efficient. Some coumarins possess antiplatelet activity and, due to their additional antioxidant effects, may be promising drugs for use in combination with the present therapeutic agents. The aim of this study was to analyse a series of simple 4-methylcoumarins for their antiplatelet activity. Human plasma platelet suspensions were treated with different aggregation inducers [arachidonic acid (AA), collagen and ADP] in the presence of the 4-methylcoumarins. Complementary experiments were performed to explain the mechanism of action. 5,7-Dihydroxy-4-methylcoumarins, in particular those containing a lipophilic side chain at C-3, reached the activity of acetylsalicylic acid on AA-induced aggregation. Other tested coumarins were less active. Some of the tested compounds mildly inhibited either collagen- or ADP-induced aggregation. 5,7-Dihydroxy-4-methylcoumarins did not interfere with the function of thromboxane synthase, but were competitive antagonists of thromboxane A(2) receptors and inhibited cyclooxygenase-1 as well. 5,7-Dihydroxy-4-methylcoumarins appear to be promising candidates for the extension of the current spectrum of antiplatelet drugs.

In vitro interactions of coumarins with iron.

Coumarins are a large group of natural substances with diverse pharmacological properties that may predetermine some of them for the prevention and/or treatment of cardiovascular diseases and also other pathologies. Free iron participates in the production of reactive oxygen species (ROS) and plays an important role in the pathogenesis of cardiovascular diseases. Therefore, chelation of iron may attenuate some ROS consequences, but on the other hand, reduction of ferric ions to ferrous ones is unfavourable and leads to intensification of ROS production. In this study, we have examined the interaction of iron with coumarins which has been rarely analyzed. A series of naturally occurring and chemically synthesized 4-methylcoumarins were analyzed for their ferrous and total iron-chelating properties and compared with standard iron chelator deferoxamine. The iron chelation activity was assessed by a simple spectrophotometric approach based on the specific indicator for ferrous ions--ferrozine. The methodology was also extended for the measurement of total iron. Among the tested coumarins, ortho-dihydroxyderivatives were the most potent iron chelators and 7,8-dihydroxy-4-methylcoumarin even reached the efficiency of deferoxamine in neutral pH. However, these ortho-dihydroxycoumarins did not bind iron firmly in acidic conditions (e.g., in acute myocardial infarction) and, moreover, they reduced ferric ions that could lead to intensification of the Fenton chemistry. Other tested coumarins did not substantially chelate iron with the exception of ortho-diacetoxycoumarins. Conclusively, the use of iron-chelating coumarins in acidic conditions may be disadvantageous in contrast to neutral conditions.

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation.

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).

Evaluation of natural substances from Evolvulus alsinoides L. with the purpose of determining their antioxidant potency.

In recent years, great attention has been given to the search for natural compounds or extracts with the purpose of medical use. Evolvulus alsinoides L. (Convolvulaceae) is a plant used in traditional medicine of East Asia in many indications and has known nootropic and anti-inflammatory activity. However, the bioactive constituents have been described poorly in the literature. Four substances isolated from the ethanol extract of E. alsinoides by means of polyamide and Silica-gel chromatography are reported here. Their molecular structures were determined using NMR analyses. There were identified as scopoletin, umbelliferone, scopolin and 2-methyl-1,2,3,4-butanetetrol. The quantity of these substances was determined using HPLC-UV and GC-FID detection. Antioxidant activity of the isolated substances was measured by DPPH assay using the SIA method. Antioxidant activity and total phenolic content of the prepared fractions are also described. The prepared fractions and isolated substances did not exhibit any significant activity in DPPH test.

Condensed and hydrolysable tannins as antioxidants influencing the health.

Natural polyphenols are a wide class of secondary plant metabolites and represent an abundant antioxidant component of human diet. An important, but often neglected group of natural polyphenols, are tannins. This review offers a general description of chemistry of both hydrolysable and condensed tannins (proanthocyanidins), the mechanisms of their antioxidation action, like free radical scavenging activity, chelation of transition metals, inhibition of prooxidative enzymes and lipid peroxidation. The mechanisms of action of antibacterial, antiviral, anticarcinogenic, cardiovascular system preventing, and antiinflammatory effects as well as the absorption, metabolic fate and positive in vivo effects of tannins are enclosed.

Evaluation of natural antioxidants of Leuzea carthamoides as a result of a screening study of 88 plant extracts from the European Asteraceae and Cichoriaceae.

In recently, there has been a great interest in natural antioxidants as bioactive components of food, nutraceuticals or potential drugs against several diseases. In our study, 88 extracts from various parts of plants from European Asteraceae and Cichoriaceae were assayed for radical scavenging activity by means of DPPH (1,1-diphenyl-2-picryl hydrazyl radical) test using the SIA (Sequential injection analysis) method developed for this purpose in our laboratory. DPPH radical scavenging activity of all tested plant extracts was evaluated according to the IC(50) parameter. 29 extracts exhibited IC(50) value lower than 0.1 mg/mL. The leaves of Leuzea carthamoides (IC(50) = 0.046 mg/mL) were chosen as the most promising sample for a subsequent phytochemical study, which resulted in isolation of seven natural compounds, namely, 4',5,7-trihydroxy-6-methoxyflavone (hispidulin) (1), 5, 7, 3', 4'- tetrahydroxyflavanone (eriodictyol) (2), 3',4',5,7-pentahydroxy-6-methoxyflavonol (patuletin) (3), eriodictyol-7-beta-glucopyranoside (4), 6-hydroxykaempferol-7-O-(6''-O-acetyl-beta-D-glucopyranoside) (5), 4-hydroxybenzoic acid (6) and 3,4-dihydroxybenzoic acid (protocatechuic acid) (7). Antioxidant activity of the isolated compounds was evaluated by DPPH test and ferric reducing antioxidant power (FRAP) test and compared with trolox and quercetin. Both tests evaluated the flavonoid (5) as the most active antioxidant. This result was confirmed by comparison with known data concerning the structure/activity relationships of flavonoids.

High-performance liquid chromatography analysis of four Leuzea carthamoides flavonoids.

A simple and sensitive high-performance liquid chromatography method for the determination of four Leuzea carthamoides flavonoids, namely eriodictyol, patuletin, eriodictyol-7-beta-glucopyranoside, and 6-hydroxykaempferol-7-O-(6"-O-acetyl-beta-D-glucopyranoside), is presented. Using this method, quantitative composition of flavonoids ranged from 0.011% to 0.574% in dried plant material was determined. This method could be used in the future for the quantitative evaluation of major phenolic compounds in L. carthamoides.

In vitro antiplatelet activity of flavonoids from Leuzea carthamoides.

Plants and their secondary metabolites, including flavonoids, exhibit a wide range of biological effects. Consequently, natural substances are receiving an increased attention in medicinal research. Owing to these facts, in vitro antiplatelet activity of ethanol summary extract and four flavonoids from Leuzea carthamoides was determined in human platelet-rich plasma. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL), and thrombin were used as agonists of platelet aggregation. The summary extract showed a significant inhibition of the aggregation induced by COL and ADP. Of the tested flavonoids, eriodictyol (1) and patuletin (2) influenced COL- and AA-induced aggregation. Their IC(50) values are presented. Flavonoid glycosides eriodictyol-7-beta-glucopyranoside (3) and 6-hydroxykaempferol-7-O-(6''-O-acetyl-beta-D[small cap]-glucopyranoside) (4) were found to be weak antiplatelet agents. These results confirmed the fact that glucosylation decreases the antiplatelet activity. Quantitative composition of tested flavonoids in L. carthamoides extract was also determined. Though two of the tested flavonoids inhibited platelet aggregation, further evaluation of L. carthamoides, in order to discover other antiplatelet active compounds and possible adverse health effects, is needed.

Immune development in jejunal mucosa after colonization with selected commensal gut bacteria: a study in germ-free pigs.

The immunological structure of the porcine jejunal lamina propria in germ-free piglets was compared with that of their counterparts associated with two strains of commensal Escherichia coli, A0 34/86 serotype O83:K24:H31 and the O86 E. coli strain, up to 20 days post-colonization. In the antigen-presenting compartment, both dendritic cells (DC) and cells expressing CD163, probably macrophages were investigated. In addition we also assessed the number of CD2+/CD3+ (T) cells. In contrast to some previous reports, we show a total lack of both DC and T cells for germ-free animals in the diffuse lymphoid tissue of villi and crypts of the jejunum. Association with either strain of commensal E. coli had a profound effect on the immune structure and resulted in extensive recruitment of DC to the lamina propria and of T cells to epithelium and lamina propria. The data suggest that the earliest immigrant cells were monocytes, which soon acquired the phenotype of mucosal DC. T cells migrated in at a slightly slower rate. Nevertheless, the response could be extremely rapid: within 3 days of colonization with O83, the magnitude of this response was comparable to that observed 20 days post-colonization.

Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells.

BACKGROUND: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RB(high) subset of CD4+T cells isolated from the spleen of normal BALB/c mice. METHODS: A CD4+CD45RB(high) subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ-free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen-free (SPF) bacteria. Mice were evaluated 8-12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). RESULTS: After the transfer of the CD4+CD45RB(high) T-cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO-1 (zona occludens). CONCLUSIONS: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB.

Decrease of dehydrogenase activity of cerebral glyceraldehyde-3-phosphate dehydrogenase in different animal models of Alzheimer's disease.

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the beta-amyloid precursor protein (betaAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the different animal models of AD: transgenic mice (Tg2576) and rats treated with beta-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon gamma (INFgamma). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of beta-amyloid, or thiorphan, an inhibitor of neprilysin involved in beta-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFNgamma, which can influence the progression of the AD, significantly reduced the enzyme activity.

HlyA knock out yields a safer Escherichia coli A0 34/86 variant with unaffected colonization capacity in piglets.

Escherichia coli A0 34/86 (O83:K24:H31) has been successfully used for prophylactic and therapeutic intestinal colonization of premature and newborn infants, with the aim of preventing nosocomial infections. Although E. coli A0 34/86 was described as a nonpathogenic commensal, partial sequencing revealed that its genome harbours gene clusters highly homologous to virulence determinants of different types of E. coli, including closely linked genes of the alpha-haemolysin operon (hlyCABD) and for the cytotoxic necrotizing factor (cnf1). A haemolysin-deficient mutant (Delta hlyA) of E. coli A0 34/86 was generated and its colonization capacity was determined. The results show that a single dose of the A0 34/86 wild-type or Delta hlyA strains resulted in efficient intestinal colonization of newborn conventional piglets, and that this was still considerable after several weeks. No difference was observed between the wild-type and the mutant strains, showing that haemolysin expression does not contribute to intestinal colonization capacity of E. coli A0 34/86. Safety experiments revealed that survival of colostrum-deprived gnotobiotic newborn piglets was substantially higher upon colonization by the nonhaemolytic strain than following inoculation by its wild-type ancestor. We suggest that the E. coli A0 34/86 Delta hlyA mutant may represent a safer prophylactic and/or immunomodulatory tool with unaffected colonization capacity.

Effect of bacterial monoassociation on brush-border enzyme activities in ex-germ-free piglets: comparison of commensal and pathogenic Escherichia coli strains.

This study was designed to investigate the effect of monoassociation of germ-free piglets with Escherichia coli strains on the development of intestinal brush-border enzyme activities. Piglets were delivered by hysterectomy, reared for seven days under germ-free conditions and fed milk formula diet. One group was maintained germ-free, the other four groups were monoassociated on day eight with one of four E. coli strains: non-pathogenic O86 or O83 and G58-1, or pathogenic 933D. The development of brush-border digestive enzyme functions in the small intestine was evaluated after 15 days. Germ-free controls exhibited slower developmental declines of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and delayed increases of sucrase and glucoamylase compared to conventionally grown animals. Association of germ-free piglets with the non-pathogenic E. coli strains O86 and O83 resulted in increased enterocyte differentiation along the length of the small intestine, accompanied by declining activities of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and elevated activities of maturational markers such as sucrase and glucoamylase. Maturational changes also occurred along the villus-crypt axis, as revealed by histochemical localization of aminopeptidase N on the villi tips in piglets colonized with E. coli O83. Interestingly, colonization with the pathogenic E. coli strain 933D stimulated changes in the main differentiation enzyme markers lactase, sucrase and glucoamylase to an extent comparable with those produced by the non-pathogenic and probiotic E. coli strains. In conclusion, germ-free piglets represent a valuable tool to study the consequences of colonization of the immature sterile gut with defined strains of bacteria.

Prenatal development of the porcine TCR delta repertoire: dominant expression of an invariant T cell receptor Vdelta3-Jdelta3 chain.

The prenatal development of the porcine gamma/delta TCR repertoire was studied by complementarity-determining region 3 (CDR3) spectratyping and sequencing of TRDV1-DV5 transcripts. Specimens from the small and large intestine, spleen, thymus, liver, bone marrow and PBMC from fetal piglets between 38 and 114 days of gestation (DG) were examined. The TCR delta repertoire was highly restricted early in gestation (DG38-DG57) and an invariant TRDV3 transcript, lacking the N/D region, was found in different fetuses throughout gestation and dominated the TRDV3 repertoires of all organs at mid gestation ( approximately DG55). Near the end of gestation, this invariant TRDV3 transcript was absent from the thymus but was still present, in a less dominant manner, in the intestine and spleen. The average CDR3 length of all Vdelta subgroups increased with ontogeny, suggesting an increase in activity of TdT. Thus, the persistence of fetal gamma/delta T cells expressing an invariant TRDV3 chain throughout development is especially surprising since TdT is active early in gestation in swine. We speculate that these gamma/delta T cells might have been selectively expanded by (self)-ligands and may have an important function throughout fetal development.

Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases.

Commensal microflora (normal microflora, indigenous microbiota) consists of those micro-organisms, which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). The number of bacteria colonising mucosal and skin surfaces exceeds the number of cells forming human body. Commensal bacteria co-evolved with their hosts, however, under specific conditions they are able to overcome protective host responses and exert pathologic effects. Resident bacteria form complex ecosystems, whose diversity is enormous. The most abundant microflora is present in the distal parts of the gut; the majority of the intestinal bacteria are Gram-negative anaerobes. More than 50% of intestinal bacteria cannot be cultured by conventional microbiological techniques. Molecular biological methods help in analysing the structural and functional complexity of the microflora and in identifying its components. Resident microflora contains a number of components able to activate innate and adaptive immunity. Unlimited immune activation in response to signals from commensal bacteria could pose the risk of inflammation; immune responses to mucosal microbiota therefore require a precise regulatory control. The mucosal immune system has developed specialised regulatory, anti-inflammatory mechanisms for eliminating or tolerating non-dangerous, food and airborne antigens and commensal micro-organisms (oral, mucosal tolerance). However, at the same time the mucosal immune system must provide local defense mechanisms against environmental threats (e.g. invading pathogens). This important requirement is fulfilled by several mechanisms of mucosal immunity: strongly developed innate defense mechanisms ensuring appropriate function of the mucosal barrier, existence of unique types of lymphocytes and their products, transport of polymeric immunoglobulins through epithelial cells into secretions (sIgA) and migration and homing of cells originating from the mucosal organised tissues in mucosae and exocrine glands. The important role of commensal bacteria in development of optimally functioning mucosal immune system was demonstrated in germ-free animals (using gnotobiological techniques). Involvement of commensal microflora and its components with strong immunoactivating properties (e.g. LPS, peptidoglycans, superantigens, bacterial DNA, Hsp) in etiopathogenetic mechanism of various complex, multifactorial and multigenic diseases, including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, multiorgan failure, colon cancer has been recently suggested. Animal models of human diseases reared in defined gnotobiotic conditions are helping to elucidate the aetiology of these frequent disorders. An improved understanding of commensal bacteria-host interactions employing germ-free animal models with selective colonisation strategies combined with modern molecular techniques could bring new insights into the mechanisms of mucosal immunity and also into pathogenetic mechanisms of several infectious, inflammatory, autoimmune and neoplastic diseases. Regulation of microflora composition (e.g. by probiotics and prebiotics) offers the possibility to influence the development of mucosal and systemic immunity but it can play a role also in prevention and treatment of some diseases.

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs.

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.

Mucosal immunity: its role in defense and allergy.

The interface between the organism and the outside world, which is the site of exchange of nutrients, export of products and waste components, must be selectively permeable and at the same time, it must constitute a barrier equipped with local defense mechanisms against environmental threats (e.g. invading pathogens). The boundaries with the environment (mucosal and skin surfaces) are therefore covered with special epithelial layers which support this barrier function. The immune system, associated with mucosal surfaces covering the largest area of the body (200-300 m(2)), evolved mechanisms discriminating between harmless antigens and commensal microorganisms and dangerous pathogens. The innate mucosal immune system, represented by epithelial and other mucosal cells and their products, is able to recognize the conserved pathogenic patterns on microbes by pattern recognition receptors such as Toll-like receptors, CD14 and others. As documented in experimental gnotobiotic models, highly protective colonization of mucosal surfaces by commensals has an important stimulatory effect on postnatal development of immune responses, metabolic processes (e.g. nutrition) and other host activities; these local and systemic immune responses are later replaced by inhibition, i.e. by induction of mucosal (oral) tolerance. Characteristic features of mucosal immunity distinguishing it from systemic immunity are: strongly developed mechanisms of innate defense, the existence of characteristic populations of unique types of lymphocytes, colonization of the mucosal and exocrine glands by cells originating from the mucosal organized tissues ('common mucosal system') and preferential induction of inhibition of the responses to nondangerous antigens (mucosal tolerance). Many chronic diseases, including allergy, may occur as a result of genetically based or environmentally induced changes in mechanisms regulating mucosal immunity and tolerance; this leads to impaired mucosal barrier function, disturbed exclusion and increased penetration of microbial, food or airborne antigens into the circulation and consequently to exaggerated and generalized immune responses to mucosally occurring antigens, allergens, superantigens and mitogens.