NO Production In RAW Research Articles

Three New Sesquiterpenoids and One New Sesquiterpenoid Derivative from Chinese Eaglewood.

Three new sesquiterpenoids (1–3) and one new sesquiterpenoid derivative (4), along with three known sesquiterpenoids (5–7), were isolated from the 95% ethanolic extract of Chinese eaglewood [Aquilaria sinensis (Lour.) Gilg]. The structures of these compounds were elucidated through extensive analysis of spectroscopic data including IR, NMR, HRESIMS, and X-ray diffraction experiments. In addition, the above new compounds were detected for their bioactivities against LPS-induced NO production in RAW 264.7 cells. Among them, compound 2 exhibited obvious anti-inflammatory activity with an IC50 value of 8.1 μM.

Clerodane-type diterpenoids from tuberous roots of Tinospora sagittata (Oliv.) Gagnep

Three new clerodane type diterpenoids (2–4), together with one known analogues (1), were isolated from the tuberous roots of Tinospora sagittata (Oliv.) Gagnep. Compound 3 is an unusual clerodane diterpenoid with the carbonyl functionality at C-3, which represents the first example from this diterpenoid compounds class. The structures were established on the basis of spectroscopic data interpretation. The absolute configuration of 1 was determined for the first time by single-crystal X-ray diffraction analysis with CuKα irradiation. These isolates were evaluated for their cytotoxic activities against five human cancer cell lines and inhibitory activities on LPS-induced NO production in RAW264.7 cells.

Protective effect of Tremella fuciformis Berk extract on LPS-induced acute inflammation via inhibition of the NF-κB and MAPK pathways.

Tremella fuciformis Berk (TFB) has long been used as a traditional medicine in Asia. Although TFB exhibits antioxidant and anti-inflammatory effects, the mechanisms of action responsible have remained unknown. We confirmed the anti-inflammatory effects of Tremella fuciformis Berk extract (TFE) in RAW 264.7 cells and observed significantly suppressed LPS-induced iNOS/NO and COX-2/PGE2 production. TFE also suppressed LPS-induced IKK, IkB, and p65 phosphorylation, as well as LPS-induced translocation of p65 from the cytosol. Additionally, TFE inhibited LPS-induced phosphorylation of MAPKs. In an acute inflammation study, oral administration of TFE significantly inhibited LPS-induced IL-1β, IL-6 and TNF-α production and iNOS and COX-2 expression. The major bioactive compounds from TFB extract were identified as gentisic acid, protocatechuic acid, 4-hydroxybenzoic acid, and coumaric acid. Among these compounds, protocatechuic acid showed the strongest inhibitory effects on LPS-induced NO production in RAW 264.7 cells. Overall, these results suggest that TFE is a promising anti-inflammatory agent that suppresses iNOS/NO and COX-2/PGE2 expression, as well as the NF-κB and MAPK signaling pathways.

Phenolic Constituents and Their Anti-inflammatory Activity fromEchinochloa utilisGrains

− Seven phenolic compounds including p-coumaric acid (1), 4-hydroxybenzoic acid (2), 4-hydroxybenzaldehyde (3), vanillic acid (4), luteolin (5), acacetin (6), and tricin (7), were isolated from the methylene chloride and ethyl acetate fractions of Echinochloa utilis grains. Compounds (1 4, 6) were isolated for the first time from this plant. These compounds were tested for inhibitory activities against LPS-induced NO production in RAW 264.7 cells. Compounds 5 and 6 displayed significant inhibitory effects, with IC50 values of 27.9 ± 2.6 and 14.0 ± 1.1 μM, respectively. The results suggested that E. utilis ethanolic extract may be used as a potential source of anti-inflammatory agents and functional foods for the treatment of allergic diseases.

Chemical Constituents of Euonymus glabra

One new phenolic compound (1) and one new flavan (2), together with eight known compounds (3-10) were isolated from the stems and twigs of Euonymus glabra Roxb. Their structures were elucidated mainly on the basis of 1D and 2D spectroscopic methods and circular dichroism analysis. In addition, compounds 1-10 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages. Compounds 1-5 and 7 exhibited moderate inhibitory activities with IC50 values ranged from 5.1 to 11.9 μM.

Ent-Abietane-type diterpenoids from the roots of Euphorbia ebracteolata with their inhibitory activities on LPS-induced NO production in RAW 264.7 macrophages.

Ten ent-abietane diterpenoids (1-10), including four new (1-4) and six known ones (5-10) were isolated from the roots of Euphorbia ebracteolata. Their structures were determined by 1D, 2D NMR, and HRESIMS. Compounds 2, 4, and 7 exhibited significant inhibitory activities on lipopolysaccharide (LPS)-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 0.69, 1.97, and 0.88μM, respectively. A primary structure-activity relationship was also discussed.

Hit-to-lead optimization of phenylsulfonyl hydrazides for a potent suppressor of PGE2 production: Synthesis, biological activity, and molecular docking study.

Preliminary hit-to-lead optimization of a novel series of phenylsulfonyl hydrazide derivatives, which were derived from the high throughput screening hit compound 1 (IC50=5700nM against PGE2 production), for a potent suppressor of PGE2 production is described. Subsequent optimization led to the identification of the potent lead compound 8n with IC50 values of 4.5 and 6.9nM, respectively, against LPS-induced PGE2 production and NO production in RAW 264.7 macrophage cells. In addition, 8n was about 30- and >150-fold more potent against mPGES-1 enzyme in a cell-free assay (IC50=70nM) than MK-886 and hit compound 1, respectively. Molecular docking suggests that compound 8n could inhibit PGE2 production by blocking the PGH2 binding site of human mPGES-1 enzyme.

Anti-tumor and immunomodulatory activity of selenium (Se)-polysaccharide from Se-enriched Grifola frondosa

A polysaccharide termed Se-GP11 was extracted and purified from Se-enriched Grifola frondosa in our previous study. This study investigated the characterization, anti-tumor and immunomodulatory activity of Se-GP11. The results showed that Se-GP11 was composed of mannose, glucose and galactose with a molar ratio of 1:4.91:2.41. The weight-average molecular weight (Mw) and weight-average mean square radius (Rw) of Se-GP11 in 0.1M sodium chloride solution were 3.3×104Da and 32.8nm. Se-GP11 existed as a globular conformation with random coil structure. Se-GP11 had no anti-tumor activity against HepG-2 cells in vitro, and it significantly inhibited the growth of Heps tumor in vivo. Se-GP11 increased the relatively thymus and spleen weights as well as serum necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) levels. In addition, Se-GP11 promoted the phagocytosis and NO production of RAW264.7 as compared with that of the normal control group. The results revealed that the Se-GP11 may exhibit the anti-tumor through improving immunologic function of the tumor bearing mice.

Soluble DPP-4 up-regulates toll-like receptors and augments inflammatory reactions, which are ameliorated by vildagliptin or mannose-6-phosphate

ObjectiveStudies have shown that dipeptidyl peptidase-4 (DPP-4) inhibitors have anti-inflammatory effects. Soluble DPP-4 (sDPP-4) has been considered as an adipokine of which actions need to be further characterized. MethodsWe investigated the pro-inflammatory actions of sDPP-4 and the anti-inflammatory effects of DPP-4 inhibition, using vildagliptin, as an enzymatic inhibitor, and mannose-6-phosphate (M6P) as a competitive binding inhibitor. ResultsIn lipopolysaccharide (LPS)-stimulated RAW264.7 cells, vildagliptin suppressed the increased expression of inducible nitric oxide synthase (iNOS) and phosphorylated JNK (pJNK), activation of the NF-κB pathway, and the resultant NO and proinflammatory cytokine production. Although sDPP-4 alone did not affect the protein level of iNOS or pJNK or the production of NO in RAW264.7 cells, it did amplify iNOS expression, NO responses, and proinflammatory cytokine production in LPS-stimulated RAW264 cells. As a probable mechanism, we found that sDPP-4 caused dose-dependent increases in the expression levels of toll-like receptor 4 (TLR4) and TLR2 in RAW264.7 cells, and that these alterations were inhibited by vildagliptin, M6P, or bisindolylmaleimide II, a protein kinase C inhibitor. Either vildagliptin or M6P suppressed iNOS expression and NO and cytokine production in LPS+DPP-4-co-stimulated macrophages, while combined treatment of the co-stimulated cells with both agents had increased anti-inflammatory effects compared with either treatment alone. Intravenous injection of sDPP-4 to C57BL/6J mice increased the expression of both TLRs in kidney and white adipose tissues. ConclusionOur findings suggest that sDPP-4 enhances inflammatory actions via TLR pathway, while DPP-4 inhibition with either an enzymatic or binding inhibitor has anti-inflammatory effects.

Anti-inflammatory Inositol Derivatives from the Whole Plant of Inula cappa.

Twelve new inositol derivatives, classified into myoinositol (1-6) and l-inositol (10-15) types, along with five known analogues were isolated from the whole plant of Inula cappa. The structures of the new compounds were established by extensive analysis of mass spectrometric and 1D and 2D NMR spectroscopic data. All the tested compounds showed anti-inflammatory activities against the production of NO in RAW264.7 macrophages stimulated by lipopolysaccharide, with IC50 values ranging from 7 to 23 μM.

Triterpenoids and Flavonoids from the Leaves of Astragalus membranaceus and Their Inhibitory Effects on Nitric Oxide Production.

Four new cycloartane triterpenes, named huangqiyegenins V and VI and huangqiyenins K and L (1-4, resp.), together with nine known triterpenoids, 5-13, and eight flavonoids, 14-21, were isolated from a 70%-EtOH extract of Astragalus membranaceus leaves. The structures of the new compounds were elucidated by detailed spectroscopic analyses, and the compounds were identified as (9β,11α,16β,20R,24S)-11,16,25-trihydroxy-20,24-epoxy-9,19-cyclolanostane-3,6-dione (1), (9β,16β,24S)-16,24,25-trihydroxy-9,19-cyclolanostane-3,6-dione (2), (3β,6α,9β,16β,20R,24R)-16,25-dihydroxy-3-(β-D-xylopyranosyloxy)-20,24-epoxy-9,19-cyclolanostan-6-yl acetate (3), and (3β,6α,9β,16β,24E)-26-(β-D-glucopyranosyloxy)-16-hydroxy-3-(β-D-xylopyranosyloxy)-9,19-cyclolanost-24-en-6-yl acetate (4). All isolated compounds were evaluated for their inhibitory activities against LPS-induced NO production in RAW264.7 macrophage cells. Compounds 1-3, 14, 15, and 18 exhibited strong inhibition on LPS-induced NO release by macrophages with IC50 values of 14.4-27.1 μM.

ANTIOXIDANT AND IMMUNOSTIMULATING ACTIVITY OF POLYSACCHARIDES EXTRACTED FROM CUBED-SNAILFISH (<i>Liparis tessellatus</i> Gilbert & Burke, 1912) EGGS

An optimized enzymatic extraction using protamexTM was established for polysaccharides from Liparis tesellatus eggs (PLE). Total sugars, sulfated glycosaminoglycans, uronic acids, and hexosamines content of PLE were 43.2%, 9.0%, 23.9%, and 1.73%, respectively. Superoxide radical (SOx*) scavenging activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH*) scavenging activity of PLE was tested to proof its antioxidant properties. Results on SOx* scavenging activity revealed that PLE has antioxidant activity although it’s not surpass the activity of Ascorbic acid as standard. DPPH* scavenging activity showed that PLE has activity increasing at a concentration-dependent manner. However, at 1.25 mg/ml, the anti-DPPH activities of PLE lowered, possibly due to the pro-oxidant action of the sample. PLE also has effects on NO production of RAW 264.7 cells, suggestive of their possible immunostimulating potential in the human body. On human keratinocyte (HaCaT cell line), HT29 and AGS cells lines, PLE was proofed has no toxicity and showed proliferative effects. This effect showed that PLE could have potency as wound healing promoter. An inference can be made that, PLE has indispensable antioxidant potencies as well as cell proliferating activities on specific cell lines which are deemed necessary in the field of medicine and their utilization as functional food supplements. Keywords: Antioxidant, Fish eggs, Immunostimulator, Liparis tessellatus, Polysaccharides

Pterocarpans and triterpenoids from Gueldenstaedtia verna

Two new pterocarpan glycosides (1–2), five new triterpenoids (3–7), and 13 known analogues (14–20) were isolated from the whole plants of Gueldenstaedtia verna. These new compounds (1–7) were elucidated by extensive spectroscopic techniques including 1D (1H and 13C) and 2D NMR experiments (COSY, HSQC, HMBC and NOESY), HR-ESI-MS and chemical methods. The absolute configuration of 1 was established by the optical rotation, the comparison of experimental and calculated electronic circular dichroism (ECD) spectra and an X-ray diffraction analysis. All the isolates were evaluated for their cytotoxicities against four human cancer cell lines and inhibitory activities on LPS-induced NO production in RAW264.7 cells.

Chemical properties and bioactivities of Goji (Lycium barbarum) polysaccharides extracted by different methods

Lycium barbarum fruit (Goji or wolfberry) is widely used as a functional food as well as an herb in Chinese medicine. The polysaccharides of L. barbarum (LBPs) are well-known for immunomodulation and antioxidant activities. This study evaluated the yields, properties and bioactivities of LBPs extracted from L. barbarum fruit by different methods including hot water (HWE 100 °C), ultrasonic water (UWE 30–40 °C), subcritical water (SWE 110 °C), and ultrasound-enhanced subcritical water extraction (USWE 110 °C). The total LBP yield (80 min extraction followed by ethanol precipitation) was the highest from USWE (14% w/w) and the lowest from HWE (7.6%). The LBP from USWE also showed the highest protein (27.9%) and phenolic content (5.3%, w/w), and the highest antioxidant activities (scavenging radicals by DPPH and trolox equivalent antioxidant capacity assays; ferric reducing power). It also showed the highest immunoactivities in activating the phagocytosis and NO production of RAW264.7 macrophages. USWE was proven the most efficient method for extraction of LBPs. Temperature and ultrasound were two chief factors affecting the extraction yield, chemical properties and bioactivities of LBP.

Neolignans with a Rare 2-Oxaspiro[4.5]deca-6,9-dien-8-one Motif from the Stem Bark of Cinnamomum subavenium

Two pairs of racemic spirodienone neolignans with a rare 2-oxaspiro[4.5]deca-6,9-dien-8-one motif, named (±)-subaveniumins A (1) and B (2), were isolated from the bark of Cinnamomum subavenium. The chiral separation of the (+)-1, (-)-1, (+)-2, and (-)-2 enantiomers was accomplished via high-performance liquid chromatography on a chiral column. Their structures were elucidated using single-crystal X-ray diffraction and spectroscopic analyses (UV, IR, HRESIMS, and 1D and 2D NMR). The absolute configurations of the enantiomers were determined by comparing the experimental and calculated electronic circular dichroic spectra. The (+)-1, (-)-1, (+)-2, and (-)-2 enantiomers exhibited moderate inhibitory effects against NO production in RAW264.7 mouse macrophages induced by lipopolysaccharide, with IC50 values of 17.9, 5.6, 15.1, and 4.3 μM, respectively.

Antioxidant and anti-inflammatory properties of Chinese ilicifolius vegetable (Acanthopanax trifoliatus (L) Merr) and its reference compounds

Stems and leaves of Acanthopanax trifoliatus (L) Merr (AT) are commonly used as an edible vegetable in China. The scientific basis of antioxidant and antiinflammatory activities of AT was investigated. The polyphenol contents and free radical scavenging activities of AT ethanol extracts (EAT) were determined. Chlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, and rutin were identified as major components in EAT. LPS-induced NO production in RAW264.7 cells was decreased significantly (p<0.05) after EAT treatment, compared with controls. In vivo studies indicated that EAT had more activity than chlorogenic acid per mg of dry weight for inhibition of TPA-induced ear edema. TPA-induced increases in cytokine production (TNF-α, IL-6, MMP-9, and TIMP-1) were significantly (p<0.05) reduced by EAT treatment, compared with controls. EAT exerts anti-inflammatory activities probably due, at least in part, to chlorogenic acid.

Acetyl Eburicoic Acid from Laetiporus sulphureus var. miniatus Suppresses Inflammation in Murine Macrophage RAW 264.7 Cells

The basidiomycete Laetiporus sulphureus var. miniatus belongs to the Aphyllophorales, Polyporaceae, and grows on the needleleaf tree. The fruiting bodies of Laetiporus species are known to produce N-methylated tyramine derivatives, polysaccharides, and various lanostane triterpenoids. As part of our ongoing effort to discover biologically active compounds from wood-rotting fungi, an anti-inflammatory triterpene, LSM-H7, has been isolated from the fruiting body of L. sulphureus var. miniatus and identified as acetyl eburicoic acid. LSM-H7 dose-dependently inhibited the NO production in RAW 264.7 cells without any cytotoxicity at the tested concentrations. Furthermore it suppressed the production of proinflammatory cytokines, mainly inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6 and tumor necrosis factor α, when compared with glyceraldehyde 3-phosphate dehydrogenase. These data suggest that LSM-H7 is a crucial component for the antiinflammatory activity of L. sulphureus var. miniatus.

Evidence for inhibition of nitric oxide and inducible nitric oxide synthase in Caco-2 and RAW 264.7 cells by a Maillard reaction product [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]-methanol

We have recently isolated and characterized the chemical structure of a bioactive Maillard reaction product, [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]-methanol (F3-A), from an aqueous glucose (Glc) and lysine (Lys) Maillard reaction (MR) model system. Here, we investigate further the mechanisms for anti-inflammatory effects of this product in Caco-2 and RAW 264.7 cells. The anti-inflammatory capacity of F3-A recovered from Glc-Lys MR mixture and a synthesized product were compared with those of the specific inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), and the nuclear factor-kappa B (NF-κB) inhibitor, pyrrolidine dithiocarbamate(PDTC). F3-A produced a dose-dependent inhibition of extracellularnitric oxide (NO) production and iNOS translation in Caco-2 cells induced with interferon gamma(IFN-γ) and phorbol 12-myristate 13-acetate(PMA), and these effects were more potent than those obtained with AG. Moreover, a combination of F3-A and AG to attenuate intestinal inflammation was additive. However, F3-A inhibited only intracellular NO production in RAW 264.7 cells and did not inhibit COX-2 or NF-κB in either cell line. We conclude that the anti-inflammatory properties of F3-A are cell specific, working through different mechanism between macrophages and intestinal cells.

Tricin derivatives as anti-inflammatory and anti-allergic constituents from the aerial part of Zizania latifolia

Methanol extract of Zizania latifolia was partitioned with EtOAc, n-BuOH, and H2O. From the EtOAc layers, a new flavonolignan along with a known flavone and three known flavonolignans, tricin (1), salcolin A (2), salcolin B (3), and salcolin C (4), were isolated through repeated silica gel and ODS column chromatography. The chemical structure of the new flavonolignan was determined to be tricin-4'-O-[erythro-β-guaiacyl-(7″-O-methyl)-glyceryl] ether and was named salcolin D (5) based on physicochemical and spectroscopic data, including FT-NMR and ESI-MS. All compounds were isolated for the first time from this plant. Compounds 2-5, tricin derivatives, all exhibited higher anti-inflammatory and anti-allergy activities than tricin. In particular, salcolin D (5) was shown to have the strongest inhibitory activity against LPS-induced NO production in RAW 264.7 cells as well as β-hexosaminidase release in IgE-sensitized RBL-2H3 cells. These results suggest that the presence of tricin derivatives conveys allergy and inflammation treatment ability to Z. latifolia.

Antiinflammatory and Wound Healing Effects of Caesalpinia sappan L.

Extracted compounds from Caesalpinia sappan L. were examined for the inhibitory activity against NO, PGE2 , and TNF-α productions and on associated transcription levels using RAW264.7 cells. They were also tested for their effects on wound healing using fibroblast L929 cells. Among the compounds tested, brazilin (8) was the most effective against lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells with an IC50 value of 10.3 μM, followed by sappanchalcone (2, 31.0 μM). Brazilin (8) also inhibited PGE2 and TNF-α production with IC50 values of 12.6 and 87.2 μM, respectively. The antiinflammatory mechanism of brazilin involved down regulation of the mRNA expressions of the iNOS, COX-2, and TNF-α genes in a dose-dependent manner. An ethanol (EtOH) extract of C. sappan significantly increased fibroblast proliferation, fibroblast migration, and collagen production, whereas brazilin (8) only stimulated fibroblast migration. In addition, the EtOH extract showed no acute toxicity in mice, and it was therefore safe to make use of its potent antiinflammatory and wound healing activities. Brazilin was mainly responsible for its antiinflammatory effect through its ability to inhibit the production of NO, PGE2 , and TNF-α. This study supports the traditional use of C. sappan for treatment of inflammatory-related diseases.