LPS-stimulated Murine Macrophages Research Articles

Licocoumarone isolated from Glycyrrhiza uralensis selectively alters LPS-induced inflammatory responses in RAW 264.7 macrophages

The effects of licocoumarone (LC) isolated from Glycyrrhiza uralensis were studied in LPS-stimulated RAW 264.7 macrophages. Our study demonstrated that LC dose-dependently attenuated LPS-induced NO production by down-regulating iNOS expression. Additionally, the treatment with LC inhibited LPS-induced expression of cytokines including IL-1β, IL-6 and IL-10, but not TNF-α, at both mRNA and protein levels. Similar suppressive effects of LC were observed on LPS-stimulated murine peritoneal macrophages as well. Furthermore, LC significantly reduced LPS-stimulated NF-κB activation by inhibition of IκBα degradation and p65 phosphorylation. The results from NF-κB-luc reporter gene assay further support the inhibitory effect of LC on NF-κB activation. Further studies showed that LC also interfered with the MAPKs and STAT3 signaling pathways, which are typical inflammatory signaling pathways triggered by LPS. Taken together, these results show that LC attenuates LPS-induced cytokine gene expression in RAW 264.7 macrophages through mechanisms that involve NF-κB, MAPKs and STAT3 signaling pathways, but the pattern of inhibition differs from that of a global immunosuppresant. Our study indicates that LC is a functional constituent of Glycyrrhiza uralensis with potential implications in infectious and immune-related diseases.

MicroRNA-322 inhibits inflammatory cytokine expression and promotes cell proliferation in LPS-stimulated murine macrophages by targeting NF-κB1 (p50).

Inflammation is the body’s normal self-protection mechanism to eliminate pathogens and resist pathogen invasion. The excessive inflammatory response may lead to inflammatory lesions. The mechanisms accounting for inflammation remain hazy. miRNAs have been proposed to have crucial effects on inflammation. In the present study, we reported that lipopolysaccharide (LPS)-stimulation increased the expression levels of inflammatory cytokines and the cell-cycle progression was suppressed in RAW264.7 cells. Meanwhile, the expression of miR-322 was significantly down-regulated after LPS treatment. Bioinformatics predictions revealed a potential binding site of miR-322 in 3′-UTR of NF-κB1 (p50) and it was further confirmed by luciferase assay. Moreover, both the mRNA and protein levels of NF-κB1 (p50) were down-regulated by miR-322 in RAW264.7 cells. Subsequently, we demonstrated that miR-322 mimics decrease in the expression levels of inflammatory cytokines and cell-cycle repression can be rescued following LPS treatment in RAW264.7 cells. The anti-inflammatory cytokines expression including IL-4 and IL-10 were significantly up-regulated. Furthermore, miR-322 could also promote RAW264.7 cells proliferation. These results demonstrate that miR-322 is a negative regulator of inflammatory response by targeting NF-κB1 (p50).

The citrus flavonoid naringenin confers protection in a murine endotoxaemia model through AMPK-ATF3-dependent negative regulation of the TLR4 signalling pathway

Excessive activation of the TLR4 signalling pathway is critical for inflammation-associated disorders, while negative regulators play key roles in restraining TLR4 from over-activation. Naringenin is a citrus flavonoid with remarkable anti-inflammatory activity, but the mechanisms underlying its inhibition of LPS/TLR4 signalling are less clear. This study investigated the molecular targets and therapeutic effects of naringenin in vitro and in vivo. In LPS-stimulated murine macrophages, naringenin suppressed the expression of TNF-α, IL-6, TLR4, inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2) and NADPH oxidase-2 (NOX2). Naringenin also inhibited NF-κB and mitogen-activated protein kinase (MAPK) activation. However, it did not affect the IRF3 signalling pathway or interferon production, which upregulate activating transcription factor 3 (ATF3), an inducible negative regulator of TLR4 signalling. Naringenin was demonstrated to directly increase ATF3 expression. Inhibition of AMPK and its upstream calcium-dependent signalling reduced ATF3 expression and dampened the anti-inflammatory activity of naringenin. In murine endotoxaemia models, naringenin ameliorated pro-inflammatory reactions and improved survival. Furthermore, it induced AMPK activation in lung tissues, which was required for ATF3 upregulation and the enhanced anti-inflammatory activity. Overall, this study reveals a novel mechanism of naringenin through AMPK-ATF3-dependent negative regulation of the LPS/TLR4 signalling pathway, which thereby confers protection against murine endotoxaemia.

Solid-Phase Synthesis of Cyclic Heptapeptide Stylissatin A

The present study achieves the total synthesis of a new proline-rich cyclic heptapeptide as an inhibitor of nitric oxide production in LPS-stimulated murine macrophage RAW264.7 cells, where all the amino acids are of L-configuration. A general method was described to synthesize the cyclic peptide by a two-step solid-phase/solution synthesis strategy. The linear octapeptide was assembled by standard Fmoc chemistry in the solid phase. Subsequently cyclization was achieved by the solution method. The final product was purified by preparative RP-HPLC, and its structure was identified by HR-QTOF-MS, 1H NMR, and 13C NMR.

Thunbergia alata inhibits inflammatory responses through the inactivation of ERK and STAT3 in macrophages.

Thunbergiaalata (Acanthaceae) has been used traditionally to treat various inflammatory diseases such as fever, cough and diarrhea in East African countries including Uganda and Kenya. However, systemic studies elucidating the anti-inflammatory effects and precise mechanisms of action of T.alata have not been conducted, to the best of our knowledge. To address these concerns, we explored the anti-inflammatory effects of a methanol extract of T.alata(MTA) in macrophages. Non-cytotoxic concentrations of MTA(≤300µg/ml) inhibited nitric oxide(NO) production in lipopolysaccharide(LPS)‑stimulated RAW264.7 macrophages by transcriptional regulation of inducible NO synthase in a dose-dependent manner. The expression of cyclooxygenase-2, the enzyme responsible for the production of prostaglandinE2, was unchanged by MTA at the mRNA and protein levels. MTA treatment inhibited interleukin(IL)-6 production and decreased the mRNA expression of pro‑inflammatory cytokines, including IL-6 and IL-1β. Tumor necrosis factor-α production and mRNA expression were not regulated by MTA treatment. The decreased production of inflammatory mediators by MTA was followed by the reduced phosphorylation of extracellular signal‑regulated kinase(ERK) and signal transducer and activator of transcription3(STAT3). MTA treatment had no effect on activity of other mitogen‑activated protein kinases(MAPKs), p38, c-Jun N-terminal kinase(JNK), and nuclear factor-κB(NF-κB). These results indicate that MTA selectively inhibits the excessive production of inflammatory mediators in LPS-stimulated murine macrophages by reducing the activity of ERK and STAT3, suggesting that MTA plays an important inhibitory role in the modulation of severe inflammation.

Nitric oxide inhibitory xanthones from the pericarps of Garcinia mangostana

Mangosteen (Garcinia mangostana, Clusiaceae) is called “queen of fruit” in Southeast Asia. In the current study, three dimeric xanthones, garcinoxanthones A–C, and four monomeric xanthones, garcinoxanthones D–G, together with 18 known xanthones, were isolated from the pericarps of G. mangostana, collected in Thailand. The structures of garcinoxanthones A–G were elucidated by analysis of their 1D and 2D NMR and other spectroscopic data, and their absolute configurations were determined by the CD spectra. All seven compounds were tested for nitric oxide (NO) inhibitory activity on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Garcinoxanthones B and C significantly inhibited NO production with IC50 values of 11.3 ± 1.7 and 18.0 ± 1.8 μM, respectively, which were comparable with the positive control indomethacin (IC50 3.9 ± 0.3 μM). Moreover, garcinoxanthone B suppressed inducible NO synthase expression in a dose-dependent manner. These results reveal the presence of rare dimeric xanthones in G. mangostana and their NO inhibitory effect on LPS-stimulated murine macrophage cells.

Anti-Inflammatory Potential of Gandarusa (<I>Gendarussa vulgaris</I> Nees) and Soursoup (<I>Annona muricata</I> L) Extracts in LPS Stimulated-Macrophage Cell (RAW264.7)

Inflammation is one of the important biological responses to injury. Anti-inflammatory is therefore proposed to treat both acute and chronic inflammation. Chemical compounds of various plants are widely used in treatment of inflammation. <strong>Objective:</strong> This study aims to evaluate anti-inflammatory potential of <em>G. vulgaris</em> extract (GVE) and <em>A. muricata</em> extract (AME) on LPS-stimulated murine macrophage cell line (RAW264.7). Cell viability assay to evaluate nontoxic concentration in cell line was performed with MTS assay. Parameters to determine anti-inflammatory activity between treatment group and non treated cells, were IL-1β, TNF-α, and IL-6 which was measured with Elisa, and NO level which was measured with nitrate/nitrite colorimetric assay. Both GVE and AME of 50 and 10 μg/mL showed high viability (>90%) and it was not significantly different compared to control, makes it suitable for treatment. GVE and AME of 50 μg/mL resulted low TNF-α level in RAW264.7(313.16pg/mL and 264.69 pg/mL respectively), as well as IL-1β level (903.53 pg/mL and 905.00 pg/mL respectively) and IL-6 (175.88 pg/mL and 219.13 pg/mL respectively). Whereas, GVE and AME of 75 μg/mL showed lower NO level (9.76 μM and 9.79 μM respectively) compared to untreated cells. This research revealed that GVE and AME possess the anti-inflammatory potential indicated by inhibition of inflammatory mediators including TNF-α, IL-1β, IL-6 and NO.

Total synthesis, structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A (1a) was reported to inhibit nitric oxide production in LPS-stimulated murine macrophage RAW 264.7 cells. In the current study, solid-phase total synthesis of stylissatin A was performed by using a safety-catch linker and yielded the peptide with a trans-Phe(7) -Pro(6) linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d-allo-Ile in place of l-Ile and a different site for macrocyclization. The resulting product (d-allo-Ile(2) )-stylissatin A (1c) was also found to have the trans-Phe(7) -Pro(6) peptide conformations like rotamer 1b. Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a, 1b, and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2-7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product (1a), the rotamer (1b), and (d-allo-Ile(2) )-stylissatin A (1c) exhibited significant inhibition of NO(.) . Further investigations were focused on 1b, which also inhibited proliferation of T-cells and inflammatory cytokine IL-2 production. The analogs 2-7 weakly inhibited NO(.) production, but strongly inhibited IL-2 cytokine production compared with synthetic peptide 1b. All analogs inhibited the proliferation of T-cells, with analog 7 having the strongest effect. In the analogs, the Pro(6) residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Anti-inflammatory polyphenol constituents derived from Cissus pteroclada Hayata

A new bergenin derivative, bergenin-11-O-α-d-galactopyranoside (compound 1), together with seven known polyphenolic compounds, were isolated from the stem of Cissus pteroclada Hayata. The structures of the 8 compounds were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Moreover, the in vitro anti-inflammatory effects of compounds (1–8) in LPS-stimulated murine macrophage RAW 264.7 cells were also investigated. Our results revealed that compound 1 inhibited the production of pro-inflammatory mediators NO and PGE2 and the expression of NF-κB, TNF-α, IL-1β, iNOS and COX-2.

Design and discovery of new pyrimidine coupled nitrogen aromatic rings as chelating groups of JMJD3 inhibitors.

The histone methylation on lysine residues is one of the most studied post-translational modifications, and its aberrant states have been associated with many human diseases. In 2012, Kruidenier et al. reported GSK-J1 as a selective Jumonji H3K27 demethylase (JMJD3 and UTX) inhibitor. However, there is limited information on the structure-activity relationship of this series of compounds. Moreover, there are few scaffolds reported as chelating groups for Fe(II) ion in Jumonji demethylase inhibitors development. To further elaborate the structure-activity relationship of selective JMJD3 inhibitors and to explore the novel chelating groups for Fe(II) ion, we initialized a medicinal chemistry modification based on the GSK-J1 structure. Finally, we found that several compounds bearing different chelating groups showed similar activities with respect to GSK-J1 and excellent metabolic stability in liver microsomes. The ethyl ester prodrugs of these inhibitors also showed a better activity than GSK-J4 for inhibition of TNF-α production in LPS-stimulated murine macrophage cell line Raw 264.7 cells. Taking together, the current study not only discovered alternative potent JMJD3 inhibitors, but also can benefit other researchers to design new series of Jumonji demethylase inhibitors based on the identified chelating groups.

TRPV4 Mechanosensitive Ion Channel Regulates Lipopolysaccharide-Stimulated Macrophage Phagocytosis.

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

Validation of ethnopharmacological use as anti-inflammatory of a decoction from <i>Annona muricata</i> leaves

Background : Through this work we evaluated the potential of the aqueous extract of Annona muricata L. leaves (AMAEL) to treat inflammatory conditions. The use of decoction or infusion of this important medicinal resource is still not scientifically validated. Methods : Different doses of AMAEL were assayed in carrageenan-induced inflammation and tetradecanoylphorbol acetate (TPA)-induced edema in mice, myeloperoxidase activity (MPO) in inflamed tissue, MPO released by A-23187-stimulated rat neutrophils and nitric oxide released by murine macrophages. Acute oral toxicity and cell viability of murine macrophages were also tested. Results : A single dose of 250, 500 and 1000 mg/kg of AMAEL did not show any symptoms associated with toxicity in vivo and the viability of murine macrophages was of 100% at the assayed doses. AMAEL at 250mg/kg and 500mg/kg exerted a significant edema reduction in the carrageenan inflammation model (26.82±0.02%, p<0.05 and 52.70±0.12%, p<0.001 inhibition respectively, after the first hour). The TPAinduced topical edema model showed a significantly and dose-dependently inhibition (56% and 78% at doses of 2.5 mg/ear and 5 mg/ear, respectively). The decrease in (MPO) enzyme activity in the ear homogenates assayed at 5 mg/ear were highly significant (92.5% ± 1.83 inhibition, p<0.001) and MPO was also reduced in activated rat neutrophils at 200 μg/ml (81.98% ± 1.01 inhibition, p<0.001). AMAEL considerably decreased dose-dependently nitrite production in LPS-stimulated murine macrophages, the highest inhibition was achieved at 500 μg/ml (73.18% ± 2.36, p<001). Conclusion : this study validates the ethnomedicinal use of the decoction of Annona muricata L. leaves as anti-inflammatory agent Keywords : aqueous extract; ethnomedicinal use; inflammation; myeloperoxidase; nitric oxide

Naturally occurring hydroxytyrosol derivatives: hydroxytyrosyl acetate and 3,4-dihydroxyphenylglycol modulate inflammatory response in murine peritoneal macrophages. Potential utility as new dietary supplements.

This work evaluated the effects of extra virgin olive oil (EVOO) phenols, hydroxytyrosyl acetate (2) and 3,4-dihydroxyphenylglycol (3), as well as two new acyl derivatives of 3, 4-(1,2-di(butanoyloxy)ethyl)benzene-1,2-diol (7) and 4-(1,2-di(lauroyloxy)ethyl)benzene-1,2-diol (8), on LPS-stimulated murine peritoneal macrophages in comparison with hydroxytyrosol (HTy, 1). Compounds 2, 3, 7, and 8 showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrite levels with a significant decrease on iNOS expression [2 (50 μM, 0.44 ± 0.03; 100 μM, 0.44 ± 0.01; p < 0.01); 3 (50 μM, 0.37 ± 0.03; 100 μM, 0.37 ± 0.01; p < 0.001); 7 (50 μM, 0.45 ± 0.06; p < 0.01)] . However, only 2 and 3 down-regulated COX-2 expression [2 (50 μM, 0.72 ± 0.04, p < 0.05; 100 μM, 0.54 ± 0.06, p < 0.01); 3 (50 μM, 0.56 ± 0.05, p < 0.05; 100 μM, 0.37 ± 0.04; p < 0.001)] and prevented IKBα degradation [2 (100 μM, 1.63 ± 0.14, p < 0.01); 3 (100 μM, 1.82 ± 0.09; p < 0.01)] ; the diacylated compounds 7 and 8 showed worse anti-inflammatory activity than the parent 3. In conclusion, 2 and 3 phenolic derivatives could play an important role in the anti-inflammatory effect of EVOO. The implication of this study for the nutrition and general health of the population rests in the possible use of natural HTy derivatives with better hydrophilic/lipophilic balance, thus improving its pharmacodynamic and pharmacokinetic profiles, as new dietary supplements in foods.

Anti-inflammatory effect of litsenolide B2 isolated from Litsea japonica fruit via suppressing NF-κB and MAPK pathways in LPS-induced RAW264.7 cells

The anti-inflammatory effects of compounds isolated from the Litsea japonica fruit were investigated in LPS-stimulated murine macrophage (RAW 264.7) cells. The results indicated that litsenolide (LN)B2 significantly inhibited the LPS-induced release of pro-inflammatory mediators, such as NO and PGE2. LNC2, and, on the other hand, exhibited cytotoxicity at the same concentrations at which it exhibited an inhibitory property. Therefore, only LNB2 was used for further experimentation. LNB2 reduced the LPS-induced production of pro-inflammatory cytokines. We further investigated the mechanism by which LNB2 inhibits pro-inflammatory mediators and cytokines by examining the level of NF-κB and MAPKs phosphorylation, which all serve as key components in inflammation-induced signaling pathways in RAW 264.7 cells. LNB2 inhibited the LPS-induced phosphorylation of NF-κB and MAPKs. These results suggest that LNB2 has an anti-inflammatory effect, inhibiting the production of pro-inflammatory mediators and cytokines via inhibition of NF-κB and MAPK signaling in LPS-stimulated RAW 264.7 cells.

Anti-inflammatory and analgesic effects of pyeongwisan on LPS-stimulated murine macrophages and mouse models of acetic acid-induced writhing response and xylene-induced ear edema.

Pyeongwisan (PW) is an herbal medication used in traditional East Asian medicine to treat anorexia, abdominal distension, borborygmus and diarrhea caused by gastric catarrh, atony and dilatation. However, its effects on inflammation-related diseases are unknown. In this study, we investigated the biological effects of PW on lipopolysaccharide (LPS)-mediated inflammation in macrophages and on local inflammation in vivo. We investigated the biological effects of PW on the production of inflammatory mediators, pro-inflammatory cytokines and related products as well as the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated macrophages. Additionally, we evaluated the analgesic effect on the acetic acid-induced writhing response and the inhibitory activity on xylene-induced ear edema in mice. PW showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and interleukin-1β (IL-1β). In addition, PW strongly suppressed inducible nitric oxide synthase (iNOS), a NO synthesis enzyme, induced heme oxygenase-1 (HO-1) expression and inhibited NF-κB activation and MAPK phosphorylation. Also, PW suppressed TNF-α, IL-6 and IL-1β cytokine production in LPS-stimulated peritoneal macrophage cells. Furthermore, PW showed an analgesic effect on the writhing response and an inhibitory effect on mice ear edema. We demonstrated the anti-inflammatory effects and inhibitory mechanism in macrophages as well as inhibitory activity of PW in vivo for the first time. Our results suggest the potential value of PW as an inflammatory therapeutic agent developed from a natural substance.

Anti-inflammatory effects of Dendrobium nobile derived phenanthrenes in LPS-stimulated murine macrophages.

Dendrobium nobile belongs to the Orchidaceae family and is one of the medicinal herbs used in traditional Chinese medicine as a therapeutic agent for gastrointestinal and cardiovascular diseases. In this study, we separated three phenanthrenes (ephemeranthol A (EA), 1,5,7-trimethoxyphenanthren-2-ol (TP), dehydroorchinol (DO)) from D. nobile, and compared their anti-inflammatory activities. TP is a new phenanthrene compound and its structure was determined from (1)H, (13)C NMR and HR-ESI-MS data. To analyze the anti-inflammatory activities of the phenanthrenes, Raw 264.7 cells were used, since they are immature-macrophages and easily matured by LPS stimulation. EA and DO showed anti-inflammatory activities in the activated Raw 264.7 cells. That is, we showed that EA is a potent inhibitor of the production of nitric oxide and pro-inflammatory cytokines. The inhibitory activities of phenanthrenes were found to be caused by blockage of NF-κB activation and the phosphorylation of MAP kinases in the macrophages. These results are expected to serve as a guide for future studies on the ability of phenanthrenes to inhibit acute and chronic inflammatory diseases.

Cyclovirobuxinum D suppresses lipopolysaccharide-induced inflammatory responses in murine macrophages in vitro by blocking JAK-STAT signaling pathway.

Cyclovirobuxinum D (CVB-D), an alkaloid isolated from the Chinese medicinal plant Buxus microphylla, has been found to be effective to treat cardiac insufficiency, arrhythmias and coronary heart disease. In the present study, we investigated the effects of CVB-D on the inflammatory responses in lipopolysaccharide (LPS)-stimulated murine macrophages in vitro and the underlying mechanisms. Murine macrophage cell line RAW264.7 cells were incubated in the presence of LPS (0.1 μg/mL) for 24 h. The cell viability was measured using MTT assay. The release of NO and cytokines were detected using the Griess test and ELISA, respectively. The mRNA and protein levels were determined using RT-PCR and Western blot, respectively. Reporter gene assays were used to analyze the transcriptional activity of NF-κB. Treatment of RAW264.7 cells with CVB-D (25-300 μmol/L) did not affect the cell viability. Pretreatment with CVB-D (50, 100 and 200 μmol/L) concentration-dependently decreased NO release and iNOS expression in LPS-treated RAW264.7 cells (its IC50 value in inhibition of NO production was 144 μmol/L). CVB-D also concentration-dependently inhibited the secretion and mRNA expression of IL-1β and IL-6 in LPS-treated RAW264.7 cells. Furthermore, CVB-D remarkably inhibited the phosphorylation of STAT1 and STAT3, as well as JAK2 in LPS-treated RAW264.7 cells, but did not affect the activation of NF-κB and MAPKs pathways. Pretreatment with the JAK2 specific inhibitor AG490 (30 μmol/L) produced similar effects on NO release and iNOS expression in LPS-treated RAW264.7 cells. CVB-D exerts anti-inflammatory effects in LPS-stimulated murine macrophages in vitro at least in part by blocking the JAK-STAT signaling pathway. The anti-inflammatory actions of CVB-D may contribute to its cardioprotection.

ChemInform Abstract: Stylissatin A, a Cyclic Peptide that Inhibits Nitric Oxide Production from the Marine Sponge Stylissa massa.

Abstract A new cyclic heptapeptide, stylissatin A ( 1 ), was isolated from the Papua New Guinean marine sponge Stylissa massa . Through the use of 1D and 2D NMR spectroscopic analysis, Marfey’s method, and MS/MS analysis, its structure was determined to be cyclo-[Tyr 1 –Ile 2 –Phe 3 –Pro 4 –Ile 5 –Pro 6 –Phe 7 ]. Stylissatin A inhibited nitric oxide production in LPS-stimulated murine macrophage RAW264.7 cells with an IC 50 value of 87 μM.

Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways.

Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree Olea europaea L. Phenolic compounds present in EVOO have recognized anti-oxidant and anti-inflammatory properties. However, the activity of the total phenolic fraction extracted from EVOO and the action mechanisms involved are not well defined. The present study was designed to evaluate the potential anti-inflammatory mechanisms of the polyphenolic extract (PE) from EVOO on LPS-stimulated peritoneal murine macrophages. Nitric oxide (NO) production was analyzed by the Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. Moreover, changes in the protein expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), as well as the role of nuclear transcription factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signalling pathways, were analyzed by Western blot. PE from EVOO reduced LPS-induced oxidative stress and inflammatory responses through decreasing NO and ROS generation. In addition, PE induced a significant down-regulation of iNOS, COX-2 and mPGES-1 protein expressions, reduced MAPK phosphorylation and prevented the nuclear NFκB translocation. This study establishes that PE from EVOO possesses anti-inflammatory activities on LPS-stimulated murine macrophages.

Isolation and Bioactivity Analysis of Ethyl Acetate Extract from Acer tegmentosum Using In Vitro Assay and On-Line Screening HPLC-ABTS(+) System.

The Acer tegmentosum (3 kg) was extracted using hot water, and the freeze-dried extract powder was partitioned successively using dichloromethane (DCM), ethyl acetate (EA), butyl alcohol (n-BuOH), and water. From the EA extract fraction (1.24 g), five phenolic compounds were isolated by the silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatography. Based on spectroscopic methods such as 1H-NMR, 13C-NMR, and LC/MS the chemical structures of the compounds were confirmed as feniculin (1), avicularin (2), (+)-catechin (3), (−)-epicatechin (4), and 6′-O-galloyl salidroside (5). Moreover, a rapid on-line screening HPLC-ABTS+ system for individual bioactivity of the EA-soluble fraction (five phenolic compounds) was developed. The results indicated that compounds 1 and 2 were first isolated from the A. tegmentosum. The anti-inflammatory activities and on-line screening HPLC-ABTS+ assay method of these compounds in LPS-stimulated murine macrophages were rapid and efficient for the investigation of bioactivity of A. tegmentosum.