Vera Francisco

Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-κB and MAPK activation in macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Propolis is a bee product with numerous biological and pharmacological properties, such as immunomodulatory and anti-inflammatory activities. It has been used in folk medicine as a healthy drink and in food to improve health and prevent inflammatory diseases. However, little is known about its mechanism of action. Thus, the goal of this study was to verify the antioxidant activity and to explore the anti-inflammatory properties of propolis by addressing its intracellular mechanism of action. Caffeic acid was investigated as a possible compound responsible for propolis action. MATERIALS AND METHODS The antioxidant properties of propolis and caffeic acid were evaluated by using the 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging method. To analyze the anti-inflammatory activity, Raw 264.7 macrophages were treated with different concentrations of propolis or caffeic acid, and nitric oxide (NO) production, a strong pro-inflammatory mediator, was evaluated by the Griess reaction. The concentrations of propolis and caffeic acid that inhibited NO production were evaluated on intracellular signaling pathways triggered during inflammation, namely p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK1/2), the transcription nuclear factor (NF)-κB and extracellular signal-regulated kinase (ERK1/2), through Western blot using specific antibodies. A possible effect of propolis on the cytotoxicity of hepatocytes was also evaluated, since this product can be used in human diets. RESULTS Caffeic acid showed a higher antioxidant activity than propolis extract. Propolis and caffeic acid inhibited NO production in macrophages, at concentrations without cytotoxicity. Furthermore, both propolis and caffeic acid suppressed LPS-induced signaling pathways, namely p38 MAPK, JNK1/2 and NF-κB. ERK1/2 was not affected by propolis extract and caffeic acid. In addition, propolis and caffeic acid did not induce hepatotoxicity at concentrations with strong anti-inflammatory potential. CONCLUSIONS Propolis exerted an antioxidant and anti-inflammatory action and caffeic acid may be involved in its inhibitory effects on NO production and intracellular signaling cascades, suggesting its use as a natural source of safe anti-inflammatory drugs.

Anti-Inflammatory Activity of Cymbopogon citratus Leaf Infusion in Lipopolysaccharide-Stimulated Dendritic Cells: Contribution of the Polyphenols

Cymbopogon citratus, an herb known worldwide as lemongrass, is widely consumed as an aromatic drink, and its fresh and dried leaves are currently used in traditional cuisine. However, little is known about the mechanism of action of C. citratus, namely, the anti-inflammatory effects of its dietary components. Because nitric oxide (NO), produced in large quantities by activated inflammatory cells, has been demonstrated to be involved in the pathogenesis of acute and chronic inflammation, we evaluated the effects of the infusion of dried leaves from C. citratus, as well as its polyphenolic fractions--flavonoid-, tannin-, and phenolic acid-rich fractions (FF, TF, and PAF, respectively)--on the NO production induced by lipopolysaccharide (LPS) in a skin-derived dendritic cell line (FSDC). C. citratus infusion significantly inhibited the LPS-induced NO production and inducible NO synthase (iNOS) protein expression. All the polyphenolic fractions tested also reduced the iNOS protein levels and NO production stimulated by LPS in FSDC cells, without affecting cell viability, with the strongest effects being observed for the fractions with mono- and polymeric flavonoids (FF and TF, respectively). Our results also indicated that the anti-inflammatory properties of FF are mainly due to luteolin glycosides. In conclusion, C. citratus has NO scavenging activity and inhibits iNOS expression and should be explored for the treatment of inflammatory diseases, in particular of the gastrointestinal tract.

Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: contribution of chlorogenic acid

ETHNOPHARMACOLOGICAL RELEVANCE Cymbopogon citratus (DC.) Stapf leaves infusion is used in traditional medicine for the treatment of inflammatory conditions, however little is known about their bioactive compounds. AIM OF THE STUDY Investigate the compounds responsible for anti-inflammatory potential of Cymbopogon citratus (Cy) on cytokines production induced by lipopolysaccharide (LPS) in human and mouse macrophages, and the action mechanisms involved. MATERIALS AND METHODS An essential oil-free infusion of Cy was prepared and polyphenol-rich fractions (PFs) were obtained from it by column chromatography. Chlorogenic acid (CGA) was identified, by HPLC/PDA/ESI-MS(n). The expression of cytokines, namely TNF-α and CCL5, was analyzed by real-time RT-PCR, on LPS-stimulated human macrophages. Activation of nuclear factor (NF)-κB, a master regulator of inflammation, was investigated by western blot and gene reporter assay. Proteasome activity was assessed using a fluorogenic peptide. RESULTS Cymbopogon citratus extract and its polyphenols inhibited the cytokine production on human macrophages. This supports the anti-inflammatory activity of Cy polyphenols in physiologically relevant cells. Concerning the effect on the activation of NF-κB pathway, the results pointed to an inhibition of LPS-induced NF-κB activation by Cy and PFs. CGA was identified, by HPLC/PDA/ESI-MS(n), as the main phenolic acid of the Cy infusion, and it demonstrated to be, at least in part, responsible by that effect. Additionally, it was verified for the first time that Cy and PFs inhibited the proteasome activity, a complex that controls NF-κB activation, having CGA a strong contribution. CONCLUSIONS The results evidenced, for the first time, the anti-inflammatory properties of Cymbopogon citratus through proteasome inhibition and, consequently NF-κB pathway and cytokine expression. Additionally, Cy polyphenols, in particular chlorogenic acid, were highlighted as bioactive compounds.

Cymbopogon citratus as source of new and safe anti-inflammatory drugs: Bio-guided assay using lipopolysaccharide-stimulated macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Aqueous extracts of Cymbopogon citratus (Cy) leaves are used in traditional medicine for the treatment of inflammatory conditions, however, little is known about their mechanism of action. AIM OF THE STUDY The aim of this study is to explore the anti-inflammatory properties of Cymbopogon citratus leaves and their polyphenol-rich fractions (PFs), as well its mechanism of action in murine macrophages. MATERIALS AND METHODS A lipid- and essential oil-free infusion of Cy leaves was prepared (Cy extract) and fractionated by column chromatography. Anti-inflammatory properties of Cy extract (1.115 mg/ml) and its PFs, namely phenolic acids (530 μg/ml), flavonoids (97.5 μg/ml) and tannins (78 μg/ml), were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. As inflammatory parameters, nitric oxide (NO) production was evaluated by Griess reaction, as well as effects on cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) expression and on intracellular signaling pathways activation, which were analyzed by Western blot using specific antibodies. RESULTS Cy extract inhibited iNOS expression, NO production and various LPS-induced pathways like p38 mitogen-activated protein kinase (MAPK), c-jun NH(2)-terminal kinase (JNK) 1/2 and the transcription nuclear factor (NF)-κB. The extracellular signal-regulated kinase (ERK) 1/2 and the phosphatidylinositol-3-kinase (PI3K)/Akt activation were not affected by Cy extract. Both phenolic acid- and tannin-rich fractions significantly inhibited NF-κB activation, iNOS expression and NO production but none of the PFs modulated MAPKs or PI3K/Akt activation. Neither Cy extract nor PFs affected LPS-induced COX-2 expression but LPS-induced PGE(2) production is inhibited by Cy extract and by phenolic acid-rich fraction. CONCLUSIONS Our data provide evidence that support the usage of Cymbopogon citratus leaves extract in traditional medicine, and suggest that Cy, in particular its polyphenolic compounds, could constitute a natural source of a new and safe anti-inflammatory drug.

Differential roles of PI3-Kinase, MAPKs and NF-κB on the manipulation of dendritic cell Th1/Th2 cytokine/chemokine polarizing profile

Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and modulate immune responses by their ability to prime naïve T-cells. Upon stimuli, DC experience several morphologic, phenotypic and functional changes in a process referred to as maturation. This process is crucial to the biological functions of DC since their maturation status confer them the ability to polarize distinct T-cell subsets. In this work we explored the relevance of PI3-Kinase, Mitogen-Activated Protein Kinases (MAPKs) and NF-kappaB on cytokines/chemokines and co-stimulatory molecules expression. As experimental model, we used a fetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). Morphology and ultrastructure were analyzed by confocal and electron microscopies, respectively. Levels of phosphorylated proteins were evaluated by Western blot, production of cytokines/chemokines was analyzed by protein arrays and the expression of surface molecules was evaluated by flow cytometry. The effect of specific inhibitors of the studied signaling pathways on the transcription of cytokines/chemokines and co-stimulatory molecules was accessed by Quantitative Real-Time RT-PCR. The results showed that LPS induces significant morphological and ultrastructural changes in FSDC. Western blot analysis revealed that LPS challenge promotes an early and transient activation of NF-kappaB, ERK1/2, p38 MAPK, along with a more sustained PI3 kinase/AKT activation. The co-stimulatory CD40, CD80, CD86 and antigen-presenting MHC class I and II molecules were increased and among secreted molecules, interleukin IL-6, CCL5, G-CSF, CCL2, CXCL2 were strongly up-regulated. Using a pharmacological approach we observed that LPS-induced increase of these molecules was differentially regulated by the distinct signaling pathways. Moreover, the polarizing T(h)2 cytokines/chemokines induced by LPS in FSDC were found to be positively regulated by NF-kappaB and ERK and negatively modulated by p38 MAPK. Altogether these results suggest that the use of pharmacological inhibitors to manipulate DC maturation, namely the polarizing T(h)1/T(h)2 cytokine/chemokine profile, may be useful in the development of more specific immunotherapeutic protocols.

Essential Oil of Juniperus communis subsp. alpina (Suter) Čelak Needles: Chemical Composition, Antifungal Activity and Cytotoxicity

Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria and fungi. In the present work the composition and the antifungal activity of the oils of Juniperus communis subsp. alpina (Suter) Čelak were evaluated. Moreover, the skin cytotoxicity, at concentrations showing significant antifungal activity, was also evaluated. The oils were isolated by hydrodistillation and analysed by gas chromatography and gas chromatography–mass spectrometry. Minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) were used to evaluate the antifungal activity of the oil against dermatophytes (Epidermophyton floccosum, Microsporum canis, M. gypseum, Trichophyton mentagrophytes, T. mentagrophytes var. interdigitale, T. rubrum, T. verrucosum), yeasts (Candida albicans, C. guillermondii, C. krusei, C. parapsilosis, C. tropicalis, Cryptococcus neoformans) and Aspergillus species (Aspergillus flavus, A. fumigatus, A. niger). Cytotoxicity was tested in HaCaT keratinocytes through the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Essential oil of J. communis subsp. alpina needles was predominantly composed of monoterpene hydrocarbons (78.4%), with the main compounds being sabinene (26.2%), α‐pinene (12–9%) and limonene (10.4%). Results concerning the antifungal activity demonstrated the potential of needle oil against dermatophytes, particularly for Microsporum canis and Trichophyton rubrum with MIC and MLC of 0.32 μL/mL. Furthermore, evaluation of cell viability showed no significant cytotoxicity in HaCaT keratinocytes at concentrations between 0.32 and 0.64 μL/mL. These results show that it is possible to find appropriate doses of J. communis subsp. alpina oil with both antifungal activity and a very low detrimental effect on keratinocytes. Copyright

Differential modulation of CXCR4 and CD40 protein levels by skin sensitizers and irritants in the FSDC cell line

The development of non-animal methods for skin sensitization testing is an urgent challenge. Some of the most promising in vitro approaches are based on the analysis of phenotypical and functional modifications induced by sensitizers in dendritic cell models. In this work, we evaluated, for the first time, a fetal skin-derived dendritic cell line (FSDC) as a model to discriminate between sensitizers and irritants, through analysis of their effects on CD40 and CXCR4 protein expression. The chemicals concentrations were chosen based on a slight cytotoxicity effect (up to 15%). Protein levels were evaluated by Western blot and immunocytochemistry, after stimulation with the skin sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulphate (NiSO(4)), the non-sensitizer 2,4-dichloronitrobenzene (DCNB), and the irritants sodium dodecyl sulphate (SDS) and benzalkonium chloride (BC). All sensitizers tested increased CD40 and CXCR4 levels. In contrast, irritants decreased both proteins levels, with a more pronounced effect on CXCR4. In agreement with these results, dendritic cells derived from human peripheral blood monocytes-derived dendritic cells (MoDC) showed a similar response pattern to the skin sensitizer and irritant tested, PPD and SDS, respectively. In conclusion, evaluation of CD40 and CXCR4 proteins in chemical-treated FSDC may represent a useful tool in a future in vitro test for sensitizing assessment.

Bioactivity of Fragaria vesca leaves through inflammation, proteasome and autophagy modulation.

ETHNOPHARMACOLOGICAL RELEVANCE Fragaria vesca leaves have been used in folk medicine for the treatment of several diseases, namely gastrointestinal, cardiovascular and urinary disorders, which could be related with the potential anti-inflammatory properties of the extract. This work aims to disclose the bioactivity and the underlying action mechanism of an extract from Fragaria vesca leaves in order to support its traditional uses. MATERIALS AND METHODS A hydroalcoholic extract was prepared from Fragaria vesca leaves and its anti-inflammatory potential was evaluated through inhibition of nitric oxide production and expression of several pro-inflammatory proteins in lipopolysaccharide-triggered macrophages. Nitric oxide scavenger activity was also assessed using a standard nitric oxide donor. Since numerous inflammatory proteins are tightly regulated by ubiquitination and proteasomal degradation, the putative effect of the extract on these cellular proteolytic pathways was also disclosed. The phytochemical characterization was performed by HPLC-PDA-ESI/MSn and compared with an infusion prepared according to the traditional method. RESULTS For non-cytotoxic concentrations (80 and 160µg/mL) the extract inhibited nitrite production, probably due to a direct nitric oxide scavenging. Furthermore, inhibition of proteasome activity was verified, leading to accumulation of ubiquitinated proteins. The extract also increased the conversion of the microtubule-associated protein light chain LC3-I to LC3-II, a marker of autophagy. Polyphenols, namely ellagitannins, proanthocyanidins, and quercetin and kaempferol glucuronide derivatives were identified in Fragaria vesca leaves extract. Most of the identified phenolic compounds matched with those found in traditional preparation, the infusion. CONCLUSIONS The extract has a direct nitric oxide scavenging activity giving support to the traditional use of this plant for the treatment of inflammatory disorders. Furthermore, the extract affects the proteolytic systems but its role in cancer treatment requires further studies.

Cymbopogon citratus industrial waste as a potential source of bioactive compounds.

BACKGROUND Cymbopogon citratus (Cc), commonly known as lemongrass, is a very important crop worldwide, being grown in tropical countries. It is widely used in the food, pharmaceutical, cosmetic and perfumery industries for its essential oil. Cc aqueous extracts are also used in traditional medicine. They contain high levels of polyphenols, which are known for their antioxidant and anti-inflammatory properties. Hydrodistillation of lemongrass essential oil produces an aqueous waste (CcHD) which is discarded. Therefore a comparative study between CcHD and Cc infusion (CcI) was performed to characterize its phytochemical profile and to research its antioxidant and anti-inflammatory potential. RESULTS HPLC-PDA/ESI-MS(n) analysis showed that CcI and CcHD have similar phenolic profiles, with CcHD presenting a higher amount of polyphenols. Additionally, both CcI and CcHD showed antioxidant activity against DPPH (EC50 of 41.72 ± 0.05 and 42.29 ± 0.05 µg mL(-1) respectively) and strong anti-inflammatory properties, by reducing NO production and iNOS expression in macrophages and through their NO-scavenging activity, in a dose-dependent manner. Furthermore, no cytotoxicity was observed. CONCLUSION The data of this study encourage considering the aqueous solution from Cc leaf hydrodistillation as a source of bioactive compounds, which may add great industrial value to this crop.

The Flavone Luteolin Inhibits Liver X Receptor Activation

Luteolin is a dietary flavonoid with medicinal properties including antioxidant, antimicrobial, anticancer, antiallergic, and anti-inflammatory. However, the effect of luteolin on liver X receptors (LXRs), oxysterol sensors that regulate cholesterol homeostasis, lipogenesis, and inflammation, has yet to be studied. To unveil the potential of luteolin as an LXRα/β modulator, we investigated by real-time RT-PCR the expression of LXR-target genes, namely, sterol regulatory element binding protein 1c (SREBP-1c) in hepatocytes and ATP-binding cassette transporter (ABC)A1 in macrophages. The lipid content of hepatocytes was evaluated by Oil Red staining. The results demonstrated, for the first time, that luteolin abrogated the LXRα/β agonist-induced LXRα/β transcriptional activity and, consequently, inhibited SREBP-1c expression, lipid accumulation, and ABCA1 expression. Therefore, luteolin could abrogate hypertriglyceridemia associated with LXR activation, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism, such as hepatic steatosis, cardiovascular diseases, and diabetes.