Identification and structure activity relationship of novel flavone derivatives that inhibit the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells

In an effort to identify novel anti-inflammatory compounds, a series of flavone derivatives were synthesized and biologically evaluated for their inhibitory effects on the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), representative pro-inflammatory mediators, in LPS-induced RAW 264.7 cells. Their structure-activity relationship was also investigated. In particular, we found that compound 3g displayed more potent inhibitory activities on PGE₂ production, similar inhibitory activities on NO production and less weak cytotoxicity than luteolin, a natural flavone known as a potent anti-inflammatory agent.     

Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L.

This study was designed to isolate and identify a potent inhibitory compound against nitric oxide (NO) production from the stem bark of Ulmus pumila L. Ethyl acetate fraction of hot water extract registered a higher level of total phenolics (756.93 mg GAE/g) and also showed strong DPPH (IC₅₀ at 5.6 μg/mL) and ABTS (TEAC value 0.9703) radical scavenging activities than other fractions. Crude extract and its fractions significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells indicating that they potentially inhibited the NO production in a concentration dependent manner. Based on higher inhibitory activity, the ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography and yielded seven fractions and all these fractions registered appreciable levels of inhibitory activity on NO production. The most effective fraction F1 was further purified and subjected to ¹H, ¹³C-NMR and mass spectrometry analysis and the compound was identified as icariside E₄. The results suggest that the U. pumila extract and the isolated compound icariside E₄ effectively inhibited the NO production and may be useful in preventing inflammatory diseases mediated by excessive production of NO.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophage cells by lignans isolated from Euonymus alatus leaves and twigs

The 80% methanolic extract of Euonymus alatus leaves and twigs afforded three new lignans, (−)-threo-4,9,4′,9′-tetrahydroxy-3,7,3′,5′-tetramethoxy-8-O-8′-neolignan (1), (−)-threo-4,9,4′,9′-tetrahydroxy-3,5,7,3′-tetramethoxy-8-O-8′-neolignan (2), (7R,8R,7′R)-(+)-lyoniresinol (3), together with seventeen known lignans (4-20). The structures of 1-20 were elucidated by extensive 1D and 2D spectroscopic methods including ¹H NMR, ¹³C NMR, ¹H-¹H COSY, HMQC, HMBC and NOESY. All the isolated compounds except for dilignans (19 and 20) significantly inhibited nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

Synthesis and in vitro evaluation of homoisoflavonoids as potent inhibitors of nitric oxide production in RAW-264.7 cells

Syntheses of natural homoisoflavonoids, (±)-portulacanones A–C (4, 8 and 9), portulacanone D (6), isolated from Portulaca oleracea L. (POL) and their derivatives (3, 5 and 7) have been achieved for the first time along with the synthesis of known derivatives (1 and 2) and their in vitro inhibitory effect against NO production in LPS-induced RAW-264.7 macrophages was evaluated as an indicator of anti-inflammatory activity. All the compounds tested had a concentration-dependent inhibitory effect on NO production by RAW-264.7 macrophages without obvious cytotoxicity. Compounds 3 (97.2% at 10?μM; IC₅₀?=?1.26?µM) followed by 6 (portulacanone D) (92.5% at 10?μM; IC₅₀?=?2.09?µM), 1 (91.4% at 10?μM; IC₅₀?=?1.75?µM) and 7 (83.0% at 10?μM; IC₅₀?=?2.91?µM) were the most potent from the series. This finding was further correlated with the suppressed expression of iNOS induced by LPS. Our promising preliminary results may provide the basis for the assessment of compound 3 as a lead structure for a NO production-targeted anti-inflammatory drug development and also could support the usefulness of POL as a folklore medicinal plant in the treatment of inflammatory diseases.     

Diarylheptanoids from the fruits of Amomum kravanh and their inhibitory activities of nitric oxide production

Two new diarylheptanoids with a tetrahydropyran ring, kravanhol A (1) and kravanhol B (2), along with one known diarylheptanoid renealtin A (3) were isolated from the fruits of Amomum kravanh. The structures of compounds 1 and 2 were established by analysis of spectroscopic data and their absolute configurations were determined by Mosher's method and CD experiments. Compound 2 showed inhibitory effect on nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages with an IC₅₀ value of 38.9 ± 1.8 μM.     

Synthesis of indolyl-3-acetonitrile derivatives and their inhibitory effects on nitric oxide and PGE2 productions in LPS-induced RAW 264.7 cells

Arvelexin is one of major constituents of Brassica rapa that exerts anti-inflammatory activities. Several indolyl-3-acetonitrile derivatives were synthesized as arvelexin analogs and evaluated for their abilities to inhibit NO and PGE₂ productions in LPS-induced RAW 264.7 cells. Of the indolyl-3-acetonitriles synthesized, compound 2k, which possesses a hydroxyl group at C-7 position of the indole ring and an N-methyl substituent, more potently inhibited NO and PGE₂ productions and was less cytotoxic than arvelexin on macrophage cells.     

The relationship between structural properties and activation of RAW264.7 and natural killer (NK) cells by sulfated polysaccharides extracted from Astragalus membranaceus roots

The aim of the present study to isolate the water-soluble polysaccharide from Astragalus membranaceus roots (AMP) and investigate the structural effects on RAW 264.7 murine macrophage and natural killer (NK) cells. AMP mainly consists of carbohydrates (66.2 %), proteins (11.8 %), and sulfates (18.0 %) with minor level of uronic acid (2.0 %). The structural modification was carried out by removal of protein and sulfate from AMP through the deproteination and desulfation. After deproteination (DP), the protein content was decreased from 11.8 % to 5.4 %. Similarly, the sulfate content of desulfated AMP (DS) was decreased from 18.0%–8.1%. AMP and DP could stimulate RAW264.7 cells to produce nitric oxide (NO) and up-regulate mRNA expression through NF-κB and MAPKs pathways. However, DS showed a considerably lower level of NO production than AMP and DP, suggesting that DS could not stimulate RAW264.7 cells. AMP and its derivatives significantly increased the natural killer cells (NK cell) proliferation (113.1%–128.7%) and cytotoxicity against HeLa cells (37.4%–55.5%). However, DS showed the lowest level of NK cells activation through the expression of IFN-γ, TNF-α, Granzyme-B, and NKp44. These results suggest that sulfate groups of AMP might play a crucial role in the RAW264.7 cells and NK cells activation.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Anti-inflammatory and antioxidant activities of Sargassum horneri extract in RAW264.7 macrophages

[Purpose]In this study, we investigated whether a 70% ethanolic (EtOH) extract of Sargassum horneri had antioxidant and anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophage-like RAW 264.7 cells.[Methods]The proximate composition, fatty acids, amino acids, and dietary fiber of S. horneri, various biologically active compounds, and antioxidant activity were analyzed.[Results]The DPPH and ABTS free radical scavenging activities, as well as the reduction power, of the S. horneri extract used here were significantly increased in a concentration-dependent manner. This indicates that S. horneri contains bioactive compounds, such as phenols and flavonoids, that have excellent antioxidant activity. The cellular viability and metabolic activity results confirmed that the extract had no discernible toxicity at concentrations up to 100 μg/mL. The levels of nitrites and cytokines (PGE₂, TNF-α and IL-6), which mediate pro-inflammatory effect, were significantly inhibited by treatment with either 50 or 100 μg/mL S. horneri extract, whereas that of IL-1β was significantly inhibited by treatment with 100 μg/mL of the extract. Similarly, the expression of iNOS and COX-2 proteins also decreased according to 50 or 100 μg/mL extract concentrations. NF-κB binding to DNA was also significantly inhibited by treatment with 100 μg/mL of extract.[Conclusion]These results suggest that 70% EtOH extracts of S. horneri can relieve inflammation caused by disease or high intensity exercise.     

Inhibitory constituents of the heartwood of Dalbergia odorifera on nitric oxide production in RAW 264.7 macrophages

Two new isoflavanones (1 and 13), along with 25 known compounds (2–12, 14–27), were isolated from the EtOAc-soluble fraction of the heartwood of Dalbergia odorifera by following their potential to inhibit the LPS-induced nitric oxide production in RAW 264.7 cells. The structures of the isolated compounds were established by spectroscopic data such as ¹D, ²D NMR and MS spectrometry. Among the isolated compounds, (2S)-pinocembrin (26), showed the most potent inhibitory effect with IC₅₀ value of 18.1 μM.     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of aurones and their inhibitory effects on nitric oxide and PGE2 productions in LPS-induced RAW 264.7 cells

Sulfuretin is one of major constituents of Rhus verniciflua that exerts anti-inflammatory activities. Some of aurones were synthesized as sulfuretin derivatives and evaluated for their abilities to inhibit NO and PGE₂ production in LPS-induced RAW 264.7 cells in order to reveal the relationship. Of the aurones synthesized in the present study, 2h and 2i, which possess C-6 hydroxyl group in A-ring and methoxy substituents in B-ring, more potently inhibited NO and PGE₂ production and were less cytotoxic than sulfuretin.     

Bioactive compounds from liverworts: Inhibition of lipopolysaccharide-induced inducible NOS mRNA in RAW 264.7 cells by Herbertenoids and Cuparenoids

The inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) by herbertenoids and cuparenoids isolated from liverworts in RAW 264.7 macrophages was evaluated. Among compounds tested, herbertenediol, cuparenediol, 1,2-diacetoxyherbertene and 2-hydroxy-4-methoxycuparene exhibited significant activity. For 2-hydroxy-4-methoxycuparene, chosen as representative compound, the strong inhibitory activity was related to the inhibition on LPS-induced iNOS mRNA. The structure-activity relationship will be discussed.     

Pyrrolidinone diterpenoid from Isodon excisus and inhibition of nitric oxide production in lipopolysaccharide-induced macrophage RAW264.7 cells

A new pyrrolidinone diterpenoid, excisusin F (1), was isolated from the aerial parts of Isodon excisus (Lamiaceae), together with four known compounds, and their structures were determined mainly by NMR (1D and 2D) and mass spectrometry. Excisusin F (1) and inflexarabdonin E (3) showed potent inhibitory effects of LPS-induced nitric oxide production in RAW264.7 cells with the IC₅₀ value of 10.4 and 3.8 μM, respectively.