Lipopolysaccharide-stimulated Nitric Oxide Production Research Articles

Two New Steroidal Saponins with Potential Anti-Inflammatory Effects from the Aerial Parts of Gnetum formosum Markgr.

Gnetum formosum Markgr., a member of the Gnetaceae family, is distributed in Vietnam. This plant remains a botanical enigma with an unexplored diversity of chemical constituents and pharmacological effects. In this study, two new steroidal saponins, namely gnetumosides A (1) and B (2), were isolated from the aerial parts of G. formosum. Their chemical structures were elucidated using spectroscopic techniques, including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and NMR, along with chemical hydrolysis and comparison with the reported literature. The potential anti-inflammatory effects of the isolated compounds were evaluated by measuring lipopolysaccharide-stimulated nitric oxide (NO) production in murine macrophage cells. Notably, compound 1 exhibited the most potent inhibitory activity (IC50 = 14.10 ± 0.75 µM), comparable to dexamethasone. Additionally, the mechanisms underlying the observed anti-inflammatory effects were investigated through molecular docking and molecular dynamics simulations on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. This study is the first to investigate the chemical constituents and pharmacological effects of G. formosum.

A new labdane-type diterpenoid from leaves of Vitex rotundifolia

Vitex rotundifolia is a folk medicinal plant that has been used to treat various diseases. We have previously found that the extract of its leaves shows cytotoxic and epithelial mesenchymal transition inhibitory effect in HHUA endometrial cells. In this study, a new labdane-type diterpenoid 1 was isolated and its structure was elucidated by NMR and CD spectroscopic analysis followed by density functional theory calculations. Compound 1 showed cytotoxicity toward HHUA cells with an IC50 value of 6.4 μM, but did not inhibit phorbol ester-induced epithelial mesenchymal transition nor lipopolysaccharide-stimulated nitric oxide production.

Anti-proliferative and anti-inflammatory eudesmanolides from the flowers of Sphagneticola trilobata (L.) Pruski

Twenty-six eudesmanolides including six undescribed compounds were isolated from the flowers of Sphagneticola trilobata (L.) Pruski. Their structures were elucidated based on the interpretation of spectroscopic techniques, NMR calculation, and DP4+ analysis. The stereochemistry of (1S,4S,5R,6S,7R,8S,9R,10S,11S)-1,4,8- trihydroxy-6-isobutyryloxy-11-methyleudesman-9,12-olide (1) was demonstrated by single crystal X-ray diffraction. All eudesmanolides were evaluated for their anti-proliferative activities against four human tumor cell lines (HepG2, HeLa, SGC-7901, and MCF-7). 1α,4β-Dihydroxy-6α-methacryloxy-8β-isobutyryloxyeudesman-9,12-olide (3) and wedelolide B (8) showed pronounced cytotoxic effects against AGS cell line with IC50 values of 1.31 and 0.89 μM, respectively. Their anti-proliferative activities against AGS cells were exerted through a dose-dependent apoptosis pathway, as verified by cell and nucleus morphological assessment, clone formation assay, and Western blot analysis. Furthermore, 1α,4β,8β-trihydroxy-6β-methacryloxyeudesman-9,12-olide (2) and 1α,4β,9β-trihydroxy-6α-isobutyryloxy- 11α-13-methacryloxyprostatolide (7) performed significant inhibitory effects on lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages with IC50 values of 11.82 and 11.05 μM, respectively. Moreover, compounds 2 and 7 could block the nuclear translocation of NF-κB and reduce the expression of iNOS, COX-2, IL-1β, and IL-6 to exert anti-inflammatory effects. This study provides evidence for the utilization of the eudesmanolides from S. trilobata as lead compounds for further research due to their cytotoxic potential.

Triterpenoids from the leaves of Abies koreana and their biological activities

Seven undescribed triterpenoids, abikoranes A–G, along with three known triterpenoids were isolated from the leaves of Abies koreana E. H. Wilson. The structures of compounds were elucidated by 1D and 2D NMR, HRMS, ECD, specific rotation, and DP4+ analysis. Abikorane A represents the second example of nor-3,4-seco-17,14-friedo-lanostane triterpenoid. Among the isolates, some compounds showed strong cytotoxic activities against some of four tested cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-116) with the values of IC50 0.89–9.62 μM, inhibited lipopolysaccharide-stimulated nitric oxide production with IC50 values of 11.57–15.16 μM, and exhibited significant nerve growth factor release effect (192.54 ± 12.33%) from C6 glioma cells.

Fatty acid acylated flavonol glycosides from the seeds of Nephelium lappaceum and their nitric oxide suppression activity

Five undescribed fatty acid esters of flavonol glycosides, nephelosides A−E, along with eight known compounds, were isolated from the seeds of Nephelium lappaceum L. The structures were elucidated by extensive analysis of spectroscopic data in combination with GC-MS analysis. Potency of compounds toward nitric oxide suppression was assessed by monitoring the inhibition of lipopolysaccharide-stimulated nitric oxide production in J744.A1 macrophage cells. Nepheloside D, kaempferol and kaempferol 7-O-α-L-rhamnopyranoside showed significant activity with IC50 values of 26.5, 11.6 and 12.0 μM, respectively.

Curcubinoyl flavonoids from wild ginseng adventitious root cultures

Wild ginseng (Panax ginseng) adventitious root cultures were prepared by elicitation using methyl jasmonate and investigated further to find new secondary metabolites. Chromatographic fractionation of wild ginseng adventitious root cultures led to the isolation of eleven compounds. The chemical structures of isolated compounds were identified as four known flavanone derivatives (1–4), one new curcubinoyl derivative, jasmogin A (5) and six new curcubinoyl-flavanone conjugates, jasmoflagins A-F (6–11) by extensive spectroscopic analysis. Newly isolated curcubinoyl derivatives showed inhibitory activity against lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. Therefore, our present study suggested that elicitor stimulated plant cell cultures might contribute to the production of new metabolites.

Two new quinones and six additional metabolites with potential anti-inflammatory activities from the roots of Juglans mandshurica

Two new quinones, 4-(5-hydroxy-1,4-dioxo-1,4-dihydronaphtha-len-3-ylamino)-butyric acid methyl ester (compound 1) and 1,3-dimethoxycarbonyl-8-hydroxy-9,10-anthraquinone (2), and six known compounds (3–8) were isolated from the roots of Juglans mandshurica Maxim., a member of the Juglandaceae family. The chemical structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and compared with data from the literature. The isolated compounds were evaluated for their ability to inhibit the production of nitric oxide, tumour necrosis factor-α, and interleukin-6 by the mouse macrophage RAW 264.7 cell line after lipopolysaccharide stimulation in vitro. We found that compounds 1–4 exhibited potent anti-inflammatory effects, as indicated by suppression of lipopolysaccharide-stimulated nitric oxide and cytokine production with 50% inhibitory concentrations between 20.09 μM and 27.63 μM. These results identify two novel quinones from J. mandshurica with potential utility as anti-inflammatory compounds.

Acetate Suppresses Lipopolysaccharide-stimulated Nitric Oxide Production in Primary Rat Microglia but not in BV-2 Microglia Cells.

To show that acetate attenuates neuroinflammatory responses in activated microglia. Dietary acetate supplementation alleviates neuroglial activation in a rat model of neuroinflammation induced by intraventricular administration of lipopolysaccharide (LPS). However, the precise mechanism(s) underlying the anti-inflammatory effect of acetate, is not fully understood. To determine whether acetate has inhibitory effects on LPS-induced neuroinflammatory responses in microglia. We examined LPS-stimulated nitric oxide (NO) production in primary rat microglia and BV-2 cells. Protein expression of inducible NO synthase (iNOS) was determined by western blot analysis. The intracellular generation of reactive oxygen species (ROS) and glutathione (GSH) were also evaluated. In primary microglia, acetate decreased LPS-stimulated NO production in a dose-dependent manner, reaching significance at greater than 10 mM, and cell viability was not affected. Acetate suppressed LPS-induced expression of iNOS protein concomitantly with the decrease in NO. The LPS-induced increase in intracellular ROS production was attenuated by acetate. In addition, acetate prevented LPS-induced reduction of GSH. Notably, such suppressive effects of acetate on NO and ROS production were not observed in BV-2 cells. These findings suggest that acetate may alleviate neuroinflammatory responses by attenuating NO and ROS production in primary microglia but not in BV-2 cells. Other: All animals received humane care, and the animal protocols used in this study were approved by the Ethics Committees for Animal Experimentation.

Anti-inflammatory effect of isopimarane diterpenoids from Kaempferia galanga.

Two new isopimarane diterpenes, 1α-hydroxy-14α-methoxyisopimara-8(9),15-diene (7) and 1α,14α-dihydroxyisopimara-8(9),15-diene (9) and eight known isopimarane diterpenes including (-)-sandaracopimaradiene (1), 6β-acetoxysandaracopimaradiene-9α-ol (2), sandaracopimaradiene-7β,9α-diol (3), sandaracopimaradiene-1α,9α-diol (4), 6β-acetoxysandaracopimaradiene-9α-ol-1-one (5), 6β-acetoxysandaracopimaradiene-1α,9α-diol (6), 6β,14α-dihydroxyisopimara-8(9),15-diene (8), and 6β,14β-dihydroxyisopimara-8(9),15-diene (10) were isolated from hexane fraction of Kaempferia galanga ethanol extract. Compounds 5, 6, 8, and 9 exerted the good anti-inflammatory effect on lipopolysaccharide-stimulated nitric oxide production from RAW264.7 cells with IC50 of 11.2, 7.7, 14.3, and 12.1 μM, respectively. These four compounds inhibited nitric oxide synthase (iNOS) mRNA expression. Compounds 5 and 6 also suppressed cyclooxygenase 2 (COX-2) mRNA expression; in addition, compound 6 had mild inhibitory effect on TNF-α mRNA. Among these compounds, 5 dramatically inhibited iNOS and COX-2 mRNA expression. The influential structures were proposed to be oxygen substitute at C-1, C-6, and α-OH at C-14.

Heat-killed Tsukamurella inchonensis reduces lipopolysaccharide-induced inflammatory responses in activated murine peritoneal macrophages

Tsukamurella inchonensis (T. inchonensis) is an aerobic species of Actinomycetales which has immunomodulatory activities when used as a suspension of killed bacilli. Here, the effects of T. inchonensis on lipopolysaccharide-induced inflammatory responses in mouse peritoneal macrophages have been examined. Peritoneal macrophages were harvested by lavaging with ice cold phosphate-buffered saline. Macrophages acquired from mice treated with different doses of T. inchonensis for seven days were cultured with 20 U/ml interferon-γ and 10 µg/ml lipopolysaccharide for in vivo assays. Nitrite levels were measured by using the diazotization method based on the Griess reaction, an indirect technique to determine nitric oxide (NO) production. T. inchonensis inhibited lipopolysaccharide-stimulated NO production in mouse peritoneal macrophages from mice previously exposed to concentrations of 108 and 5 × 10<sup>7</sup> CFU per flask. Also, T. inchonensis decreased lipopolysaccharide-induced production of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α. Thus, it can be concluded that T. inchonensis is a powerful inhibitor of lipopolysaccharide-induced NO production in activated murine macrophages, and T. inchonensis may be useful as a novel agent for chemoprevention in inflammatory diseases.

Phenolic amides from Tribulus terrestris and their inhibitory effects on nitric oxide production in RAW 264.7 cells.

A new phenolic amide, named cis-terrestriamide (7), together with ten known compounds (1-6, 8-11), were isolated from the methanolic extract of the fruits of Tribulus terrestris. The structure of 7 was elucidated on the basis of extensive analyses of 1D and 2D nuclear magnetic resonance spectroscopic and high resolution mass spectrometry data. Compounds 1, 2, 5, 6, 8, 9, and 11 exhibited inhibitory effects on the lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 cells, with IC50 values of 18.7-49.4μM.

Heme Oxygenase-1-Inducing Activity of 4-Methoxydalbergione and 4'-Hydroxy-4-methoxydalbergione from Dalbergia odorifera and Their Anti-inflammatory and Cytoprotective Effects in Murine Hippocampal and BV2 Microglial Cell Line and Primary Rat Microglial Cells.

Dalbergia odorifera T. Chen (Leguminosae) grows in Central and South America, Africa, Madagascar, and Southern Asia. D. odorifera possesses many useful pharmacological properties, such as antioxidative and anti-inflammatory activities in various cell types. 4-Methoxydalbergione (MTD) and 4'-hydroxy-4-methoxydalbergione (HMTD) were isolated from the EtOH extract of D. odorifera by several chromatography methods. The chemical structures were elucidated by nuclear magnetic resonance (NMR) and mass spectrum (MS). Anti-inflammatory and cytoprotective effects were examined using BV2 microglial cells and murine hippocampus. MTD and HMTD were demonstrated to induce heme oxygenase (HO)-1 protein levels through the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in BV2 microglial cells, while only MTD upregulated HO-1 in HT22 cells. MTD and HMTD induced HO-1 expression through JNK MAPK pathway in BV2 cells, whereas only MTD activated the ERK and p38 pathways in HT22 cells. MTD was also shown to activated MTD and HMTD suppressed lipopolysaccharide-stimulated nitric oxide (NO) and prostaglandin E2 production by inhibiting inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in a dose-dependent manner. Furthermore, MTD and HMTD attenuated pro-inflammatory cytokine productions. These anti-inflammatory effects were found to be mediated through the nuclear factor-kappa B (NF-κB) pathway. MTD exhibited neuroprotective effects on glutamate-induced neurotoxicity by promoting HO-1 in HT22 cells. The anti-inflammatory and cytoprotective effects of MTD and HMTD were partially reversed by an HO inhibitor tin protoporphyrin IX. In addition, MTD and HMTD inhibited pro-inflammatory cytokines and NF-κB pathway in primary rat microglia. These findings suggest that MTD and HMTD have therapeutic potential against neurodegenerative diseases accompanied by microglial activation and/or oxidative cellular injury.

In vitro anti-inflammatory activities of blacklip abalone (Haliotis rubra) in RAW 264.7 macrophages

ABSTRACTAbalone (Haliotis sp) has been used as a traditional functional food for many years by different cultures believing that consumption provides health benefits. We investigated the anti-inflammatory activity from blacklip abalone visceral waste. An extract was prepared from the viscera using digestion with the food grade proteases papain and bromelain followed by anion exchange chromatography (AEC). The anti-inflammatory potential of the extract and AEC fractions were investigated in comparison to a plant-derived positive control (quercetin) through the inhibition of lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 monocytes. Significant anti-inflammatory activity was observed in response to the abalone extract and the unbound AEC material over a concentration (based on collagen) range of 12–96 µg/mL. Overall, results indicated that blacklip abalone extract has anti-inflammatory activity in vitro, warranting further investigation into the bioactive constituents that may be potential anti-inflammatory therapeutics.

Prenylated Xanthones from the Roots of Cudrania tricuspidata as Inhibitors of Lipopolysaccharide-Stimulated Nitric Oxide Production.

Investigation of the CH2 Cl2 and EtOAc-soluble fractions of the roots of Cudrania tricuspidata afforded 31 compounds. The structures of the isolated compounds were determined on the basis of spectroscopic data interpretation such as 1D and 2D NMR analysis and elucidated as xanthones with prenyl moieties including four simple xanthones (1-4), five prenylated xanthones with one prenyl moiety (5-7, 20, and 21), and 22 prenylated xanthones with two prenyl moieties (8-19 and 22-31). Among them, three diprenylated xanthones named cudracuspixanthones E-G (16, 29, and 31) were first isolated in nature. Xanthones 2, 5, 6, 12, 19, 21, 22, 25, 27, and 28 showed moderate inhibition of lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 cells, with IC50 values ranging from 16.1 to 24.8 µM.

Sesquiterpenes from the roots of Lindera strychnifolia with inhibitory effects on nitric oxide production in RAW 264.7 cells

Seven new sesquiterpenes, linderolides N–T (1–7), along with nine known compounds, were isolated from roots of Lindera strychnifolia (Lauraceae). Their structures were established by extensive spectroscopic analysis. The relative and absolute configurations were determined by NOESY and CD analysis, respectively. Among the isolated compounds, two new compounds, linderolide O (2) and linderolide P (3) inhibited lipopolysaccharide-stimulated nitric oxide production in murine RAW 264.7 macrophage cells, with IC50 values of 6.3 and 9.6μM, respectively.

Effects of trophic exposure to diclofenac and dexamethasone on hematological parameters and immune response in freshwater fish.

The aim of the present study was to evaluate the effects of diclofenac and dexamethasone on hematological parameters and immune response in the fish species Hoplias malabaricus after trophic exposure. Fish were fed twice every week with Astyanax sp., which were given an intraperitoneal inoculation with diclofenac (0 μg/kg, 0.2 μg/kg, 2.0 μg/kg, or 20.0 μg/kg) or dexamethasone (0.03 μg/kg, 0.3 μg/kg, or 3.0 μg/kg). After 12 doses, the hematological parameters and lipopolysaccharide-induced nitric oxide production by head kidney monocytic lineage were evaluated. Exposed fish also received 1 mg/kg of carrageenan intraperitoneal, and cell migration to the peritoneal cavity was evaluated after 4 h. Diclofenac and dexamethasone altered the red blood cell count, as well as hematocrit and hemoglobin levels. The total blood leukocyte count decreased in all groups. A significantly reduced carrageenan-induced leukocyte migration to the peritoneal cavity, particularly of polymorphonuclear cells, was observed at all tested doses, suggesting a possible immunosuppressive effect. The basal nitric oxide synthesis of head kidney cell cultures was reduced at the highest dose of diclofenac and was increased at the highest dose of dexamethasone. The lipopolysaccharide-stimulated nitric oxide production was reduced in all treatments, thus corroborating the immunosuppressive effect. Although some fish responses were variable for different drugs, the results suggested that trophic exposure to diclofenac and dexamethasone can lead to hematological changes and immunotoxic effects, causing negative impacts in aquatic organisms.

Longan (Dimocarpus longan Lour.) inhibits lipopolysaccharide-stimulated nitric oxide production in macrophages by suppressing NF-κB and AP-1 signaling pathways

Ethnopharmacological relevanceFlower, seed, and fruit of longan (Dimocarpus longan Lour.) have been used in the Traditional Chinese Medicine (TCM) serving as a common herb in relief of swelling which can be applied in cases of inflammatory diseases. However, the scientific evidence related to their effects on inflammation especially the possible cellular and molecular mechanisms of longan need to be clarified. Aim of the studyTo evaluate the anti-inflammatory effect of the various parts of longan including flower, seed, and pulp. The mechanisms and molecular targets involved in their effects were also investigated. Materials and methodsDifferent longan extracts were analyzed for their bioactive compounds and evaluated for anti-inflammation. Corilagin, ellagic acid, and gallic acid were detected using HPLC-DAD. In vitro anti-inflammatory effect of longan extracts and their polysaccharides were examined by analyzing nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Expression of the proteins that involved in NO production was detected by Western blot. ResultsFlower extract contained the highest amounts of total phenolics, total flavonoids, proanthocyanidins, corilagin and ellagic acid when compared to seed and pulp extracts. The extracts of different longan parts inhibited LPS-induced NO production with different potency. The highest potency for the inhibition of NO production was shown with flower extract follow by seed and pulp (IC50=128.2, 1127.4, and 1260.2μgmL−1, respectively). The mechanisms of this anti-NO production were associated with their NO scavenging effect and their decreasing the expression and catalytic activity of an inducible nitric oxide synthase (iNOS). Moreover, these longan extracts suppressed LPS-induced degradation of IκBα and activation of NF-κB, activator protein-1 (AP-1), Akt, and mitogen activated protein kinases (MAPKs). ConclusionThese results suggest that the longan extracts possess anti-inflammatory property. Therefore, longan could provide potential dietary supplement for the treatment of inflammatory-related diseases.

Two new limonoids from the roots of Trichilia connaroides with inhibitory activity against nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells

Two new rearranged limonoids, trichiliton I (1) and 12-deacetoxyltrijugin A (2) along with four previously reported compounds (3–6) were isolated from the roots of Trichilia connaroides. Their structures were established on the basis of extensive spectroscopic analysis including HR-ESI–MS and 1D and 2D NMR. The new compounds 1 and 2 exhibited weak inhibitory effects on lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 cells in a dose-dependent manner.

Three new bioactive phenolic glycosides from Liparis odorata

Chemical investigation of the ethanol extract from the whole plant Liparis odorata (Willd.) Lindl (orchid family) has led to the isolation of three new phenolic glycosides named liparisglycoside A–C (1–3) along with six known compounds (4–9). The structures of compounds were elucidated by means of extensive spectroscopic data analysis and comparison with the literature data. In addition, compounds 3 and 9 revealed inhibitory effects on lipopolysaccharide-stimulated nitric oxide production, and compounds 1, 3 and 9 displayed hypolipidaemic effects in in vitro bioassays.

Korean Red Ginseng saponin fraction modulates radiation effects on lipopolysaccharide-stimulated nitric oxide production in RAW264.7 macrophage cells

BackgroundIn previous work, we reported that Korean Red Ginseng saponin fraction (RGSF) showed anti-inflammatory activities in vitro and in vivo. MethodsThe present study investigated the radioprotective properties of RGSF by examining its effects on ionizing radiation (IR)-enhanced and lipopolysaccharide (LPS)-mediated inflammatory responses in murine macrophage cells. ResultsRGSF induced strong downregulation of IR-enhanced and LPS-induced proinflammatory responses such as nitric oxide (NO) production (Inhibitory Concentration 50 (IC50) = 5.1 ± 0.8 μM) and interleukin-1β levels. RGSF was found to exert its radioprotective effects by inhibition of a signaling cascade that activated checkpoint kinase 2–nuclear factor-κB. In addition, RGSF strongly inhibited IR-enhanced LPS-induced expression of hemoxyganase-1, implying that the latter may be a potential target of RGSF. ConclusionTaken together, our data suggest that RGSF can be considered and developed for use as an effective radioprotective agent with minimal adverse effects.