NO Production In RAW Research Articles

Synthesis of novel andrographolide beckmann rearrangement derivatives and evaluation of their HK2-related anti-inflammatory activities

Two series of andrographolide derivatives with introduction of amide moiety into ring A by Beckmann rearrangement were synthesized. In series 1, the ring A was converted to caprolactam, and an amide moiety was linked to C-19 of ring A in series 2. Among them, compound 8h exhibited obvious inhibition on HK2 enzyme activity (IC50 = 9.36 ± 0.08 μM) and LPS-induced NO production in RAW264.7 cells (IC50 = 22.38 ± 3.57 μM), and potent binding affinity with HK2 (Kd = 5.12 ± 0.82 μM). The preliminary structure-activity relationships (SARs) suggested that the formation of caprolactam of ring A and esterification of C-19-hydroxyl could improve the inhibitory effects on HK2 enzyme of andrographolide derivatives. Furthermore, compound 8h significantly reduced the levels of IL-1β and IL-6, down-regulated the expressions of iNOS and COX-2. Its anti-inflammatory effect was related to the inhibition of both NF-κB pathway and glycolysis enzyme HK2. Since HK2 could be a novel and effective target for anti-inflammation, compound 8h might be a new anti-inflammatory agent targeting at HK2, or serve as a lead compound to design and synthesize more HK2 inhibitors with better inflammatory effects.

Anti-Inflammatory Effects of Grasshopper Ketone from Sargassum fulvellum Ethanol Extract on Lipopolysaccharide-Induced Inflammatory Responses in RAW 264.7 Cells.

This study evaluated the anti-inflammatory potential of a grasshopper ketone (GK) isolated from the brown alga Sargassum fulvellum on lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophage cell line. GK was isolated and purified from the n-hexane fraction and its structure was verified on the basis of NMR spectroscopic data. GK up to 100 μg/ml is not cytotoxic to RAW 264.7, and is an effective inhibitor of LPS-induced NO production in RAW 264.7 cells. The production of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α was found significantly reduced in 0.1-100 μg/ml dose ranges of GK treatment (p < 0.05). We confirmed the dose-dependent and significant inhibition of iNOS and COX-2 proteins expression. In addition, it has been shown that GK induces anti-inflammatory effects by inhibiting MAPKs (ERK, JNK, and p38) and NF-κB p65 phosphorylation. Our results show that the anti-inflammatory properties of GK may be due to the inhibition of the NF-κB and MAPKs pathways, which are associated with the attenuation of cytokine secretion.

Two new xanthones, a new lignin, and twenty phenolic compounds from Smilax china and their NO production inhibitory activities

Two new xanthones smilone A (1), smilone B (2), and a new lignin smilgnin A (3) were isolated from the rhizomes of Smilax china L., together with three known xanthones (4–6), four lignins (7–10), two flavones (11, 12), two stilbenoids (13, 14), and ten organic phenoloids (15–24). Of them, compounds 4–6 were isolated from the genus Smilax for the first time. The structures of 1–24 were elucidated by the extensive analysis of spectral data and compared with the literature. All compounds were evaluated for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages. Among them, compound 24 exhibited significant inhibitory activity against NO production (IC50 = 1.26 µM), while compounds 3, 6, and 7 showed weak activities at the concentration of 50 μM.

New acetylenic compounds and lignans from Dolomiaea berardioidea (Franch.) Shih

Two new acetylenic compounds (1 and 2), two new lignans (3 and 4) along with eight known lignans (5−12), ten known terpenoids (13−22) and one known aromatic compound (23) were isolated from the whole plants of Dolomiaea berardioidea (Franch.) Shih. Structures of new compounds were elucidated by combined use of HRESIMS, 1D and 2D NMR and CD spectroscopic methods. The absolute structures of 1 and 3 were further confirmed by Cu Kα X-ray crystallographic analyses. In addition, all isolated compounds were also tested for their inhibitory effects against LPS-induced NO production of RAW 264.7 macrophage. Compound 22 displayed potential anti-inflammatory activity with IC50 value of 7.4 μM.

Anti-inflammatory Mono- and Dimeric Sorbicillinoids from the Marine-Derived Fungus Trichoderma reesei 4670.

Eight new dimeric sorbicillinoids (1-3, 5-9) and 12 new monomeric sorbicillinoids (10-20, 25), along with five known analogues (4 and 21-24), were isolated from the marine-derived fungus Trichoderma reesei 4670. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HR-ESIMS, and ECD) and X-ray crystallography. Compound 1, containing a pyrrolidin-2-one moiety, is reported for the first time in the sorbicillinoid family. Compounds 8 and 9 are the first examples of bisorbicillinoids possessing a benzofuro[2,3- h]chromene scaffold from a natural source. Compounds 3-11, 13-16, 18, 21, 22, 24, and 25 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide with IC50 values in the range from 0.94 to 38 μM. Structure-activity relationships of the sorbicillinoids were discussed.

The polysaccharide from the roots of Actinidia eriantha activates RAW264.7 macrophages via regulating microRNA expression.

The polysaccharide from the roots of Actinidia eriantha (AEPS), a potent antitumor agent and immunological adjuvant, has been previously reported to activate RAW264.7 macrophages via TLRs/NF-κB signaling. The objective of this study was to investigate microRNA (miRNA) expression profiles and explore the role of miRNAs in AEPS-activated RAW264.7 cells using microarray assay and specific inhibitor. AEPS induced 82 differentially expressed miRNAs including 43 up-regulated and 39 down-regulated in RAW264.7 cells. A set of 11 differentially expressed miRNAs and their 62 target mRNAs were predicted to involve in activation of RAW264.7 cell induced by AEPS based on computational databases and validated reports. miR-155 inhibitor significantly down-regulated the mRNA expression of proinflammatory factors (IL-1β, IL-6, COX-2 and NOS2), and blocked the production of NO in RAW264.7 cell induced by AEPS. The expression of accessory and costimulatory molecules (CD80, CD86, MHC I and MHC II) in AEPS-activated RAW264.7 cells was also remarkably reduced by miR-155 inhibitor. These results suggested that AEPS may induce macrophage activation through regulating miRNAs expression such as miR-155. This study further expanded current knowledge on the mechanisms of plant polysaccharides.

Chloraserrtone A, a Sesquiterpenoid Dimer from Chloranthus serratus.

Chloraserrtone A (1), a new sesquiterpenoid dimer with two lindenane-type sesquiterpenoid monomers bridged by two six-membered rings, was obtained from Chloranthus serratus. A combination of UV, IR, NMR, HRESIMS, and X-ray diffraction data were used to elucidate the structure of 1. Compound 1 represents the first lindenane-type sesquiterpenoid dimer with extremely unique C-15-C-15', C-4-C-6', and C-6-C-11' linkages to form two six-membered rings between the monomeric units. A plausible biosynthesis toward chloraserrtone A is proposed. This new compound (1), together with the known lindenane dimers (2-11), were assessed for their inhibitory effects on lipopolysaccharide-induced NO production in RAW264.7 cells. Compound 6 showed activity with an IC50 value of 3.7 μM.

Caesalminaxins O-T, cassane diterpenoids from the seeds of Caesalpinia minax and their anti-inflammation

Six previously undescribed cassane diterpenoids, named caesalminaxins O-T (1–6), together with 28 known compounds (7–34), were isolated from the seeds of Caesalpinia minax Hance. Their structures, including their absolute configurations were elucidated by extensive spectroscopic analysis, X-ray diffraction, and quantum chemical calculations. Among the undescribed diterpenoids, compound 6 that possessed an unusual enol group at C-7 with a highly deshielded 1H NMR signal was the first example in cassane diterpenoids. All of the isolates were evaluated for their inhibitory activity against lipopolysaccharide-activated NO production in RAW264.7 cells. Compound 16 showed moderate inhibitory activity with an IC50 value of 17.3 μM, which was more potent than the positive control (indomethacin, IC50 = 29.7 μM).

Anti‐inflammatory and cytotoxic activities of constituents from Wedelia trilobata (L.) Hitchc.

AbstractUsing various chromatographic separations, five compounds (1–5) were isolated from a methanol extract of W. trilobata leaves. Their structures were elucidated to be paniculoside‐IV (1), 3‐O‐[β‐D‐glucopyranosyl(1‐4)‐β‐D‐glucoronopyranosyl] oleanolic acid 28‐O‐β‐D‐glucopyranosyl ester (2), 5,7,4'‐trihydroxyflavone (3), apigenin 7‐O‐β‐D‐glucopyranoside (4), and 3,4‐dihydroxy‐cinnamic acid (5), by detailed analysis via spectroscopic techniques (IR, 1D, 2D NMR and ESI‐MS data) as well as comparison with those reported. This is the first report of 2 from Wedelia genus. Furthermore, the effects of the isolated compounds on LPS‐induced NO production in RAW264.7 cells and cytotoxic activity (against A549, MCF‐7, Hep3B, and PC3 cell lines) were also evaluated.

Structures and biological evaluation of phenylpropanoid derivatives from Murraya koenigii

Four new phenylpropanoid derivatives (1–4), together with eleven known analogues (5–15) were isolated and identified by comparison with their references and extensive spectroscopic methods from Murraya koenigii for the first time. Compounds (1–15) were assayed for their inhibitory activities by measuring IL-6-induced STAT3 promoter activities in HepG2 cells, and found compounds 1, 2, 6, and 15 showed inhibitory effects with IC50 values of 11.5, 18.7, 8.9, and 22.7 μM, respectively. The inhibitory activities of compounds (1–15) were screened against NO production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells, and found compounds 3, 4, 9, 11, and 14 exhibited inhibitions against LPS-induced NO production in RAW264.7 macrophages, with IC50 values of 32.7, 7.9, 42.1, 58.9, and 62.4 μM, respectively.

Acylated flavonoid glycosides from Barringtonia racemosa

Using various chromatographic separations, three new acylated flavonoid glycosides, namely barringosides G–I (1–3), were isolated from the water-soluble extract of Barringtonia racemosa branches and leaves. The structure elucidation was performed by extensive analysis of the 1D and 2D NMR and HR-QTOF-MS data. Of the isolated compounds, barringoside I (3) showed moderate inhibitory effects on LPS-induced NO production in RAW264.7 cells with an IC50 of 52.48 ± 1.04 µM.

Subcritical water extraction as an efficient technique to isolate biologically-active fucoidans from Nizamuddinia zanardinii.

A sulfated polysaccharide (fucoidan) has been isolated from Nizamuddinia zanardinii using subcritical water extraction method (SCWE), and extraction conditions were optimised using the response surface methodology. The optimum extraction conditions were found to be: extraction time of 29 min, extraction temperature of 150 °C, and raw material-to-water ratio of 21 g/mL. The fucoidan yield under these optimum conditions was 25.98%, which was considerably higher than that of conventional solvent extraction (5.2%). Extraction time and temperature were the extraction variables that most significantly affected fucoidan yield. Chemical and monosaccharide composition, molecular weight, and the antioxidant, anticancer and immunomodulatory activities of the extract have also been investigated. The monosaccharide composition of fucoidan included fucose (34.13%), mannose (30.70%), galactose (23.19%), xylose (9.35%) and glucose (2.65%). The average molecular weight of the extracted fucoidan was 694 kDa. Antioxidant results revealed that SCWE-extracted fucoidan had appreciable ABTS radical scavenging (70.35%) and reducing power (0.182 Abs). The anticancer activity of fucoidan ranged from 24.60 to 49.46% for HeLa cells and from 23.95 to 46.78% for HepG2 cells. The NO production of RAW264.7 cells was observed to be dose-dependent, while maximum NO production was found to be 34.82 μmol at a 50 μg/mL fucoidan concentration.

Lignans from the Twigs of Litsea cubeba and Their Bioactivities.

Litsea cubeba, an important medicinal plant, is widely used as a traditional Chinese medicine and spice. Using cytotoxicity-guided fractionation, nine new lignans 1–9 and ten known analogues 10–19 were obtained from the EtOH extract of the twigs of L. cubeba. Their structures were assigned by extensive 1D- and 2D-NMR experiments, and the absolute configurations were resolved by specific rotation and a combination of experimental and theoretically calculated electronic circular dichroism (ECD) spectra. In the cytotoxicity assay, 7′,9-epoxylignans with feruloyl or cinnamoyl groups (compounds 7–9, 13 and 14) were selectively cytotoxic against NCI-H1650 cell line, while the dibenzylbutyrolactone lignans 17–19 exerted cytotoxicities against HCT-116 and A2780 cell lines. The results highlighted the structure-activity relationship importance of a feruloyl or a cinnamoyl moiety at C-9′ or/and C-7 ketone in 7′,9-epoxylignans. Furthermore, compound 11 was moderate active toward protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 13.5 μM, and compounds 4–6, 11 and 12 displayed inhibitory activity against LPS-induced NO production in RAW264.7 macrophages, with IC50 values of 46.8, 50.1, 58.6, 47.5, and 66.5 μM, respectively.

Design and synthesis of novel pyrazolo[4,3-d]pyrimidines as potential therapeutic agents for acute lung injury

Four series of total 35 new pyrazolo[4,3-d]pyrimidine compounds were designed, synthesized and evaluated for their inhibitory activity against LPS-induced NO production in RAW264.7 macrophages. Among them, compound 4e was found to be the most potent inhibitor, which decreased the production of cytokines in vitro, such as NO, IL-6 and TNF-α, with IC50 values of 2.64, 4.38 and 5.63 μM, respectively. Further studies showed that compound 4e inhibited cytokines secretion of macrophages through suppressing TLR4/p38 signaling pathway. Additionally, compound 4e showed in vivo anti-inflammatory activity in LPS-induced model of acute lung injury. These data suggested that compound 4e may be a promising lead structure for the treatment of ALI.

Synthesis and in vitro and in vivo anti-inflammatory activity of novel 4-ferrocenylchroman-2-one derivatives

A series of novel 4-ferrocenylchroman-2-one derivatives were designed and synthesised to discover potent anti-inflammatory agents for treatment of arthritis. All the target compounds had been screened for their anti-inflammatory activity by evaluating the inhibition effect of LPS-induced NO production in RAW 264.7 macrophages. Among them, 4-ferrocenyl-3,4-dihydro-2H-benzo[g]chromen-2-one (3h) was found to be the most potent compound in inhibiting the productions of NO with low toxicity. This compound also exhibited significant inhibition of the productions of IL-6 and TNF-α in RAW 264.7 macrophages. Preliminary mechanism studies indicated that compound 3h could inhibit the activation of LPS-induced NF-κB and MAPKs signalling pathways. The in vivo anti-inflammatory effect of this compound was determined in the rat adjuvant-induced arthritis model.

Sageretia thea Inhibits Inflammation through Suppression of NF- B and MAPK and Activation of Nrf2/HO-1 Signaling Pathways in RAW264.7 Cells.

Sageretia thea (S. thea) commonly known as Chinese sweet plum or Chinese bird plum has been used for treating hepatitis and fevers in Korea and China. S. thea has been reported to exert anti-oxidant, anticancer and anti-human immunodeficiency virus activity. However, there is little study on the anti-inflammatory activity of S. thea. Thus, we evaluated the anti-inflammatory effect of extracts of leaves (ST-L) and branches (ST-B) from Sageretia thea in LPS-stimulated RAW264.7 cells. ST-L and ST-B significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, COX-2, IL-1 and IL-6 in LPS-stimulated RAW264.7 cells. ST-L and ST-B blocked LPS-induced degradation of I B- and nuclear accumulation of p65, which resulted in the inhibition of NF- B activation in RAW264.7 cells. ST-L and ST-B also attenuated the phosphorylation of ERK1/2, p38 and JNK in LPS-stimulated RAW264.7 cells. In addition, ST-L and ST-B increased HO-1 expression in RAW264.7 cells, and the inhibition of HO-1 by ZnPP reduced the inhibitory effect of ST-L and ST-B against LPS-induced NO production in RAW264.7 cells. Inhibition of p38 activation and ROS elimination attenuated HO-1 expression by ST-L and ST-B, and ROS elimination inhibited p38 activation induced by ST-L and ST-B. ST-L and ST-B dramatically induced nuclear accumulation of Nrf2, but this was significantly reversed by the inhibition of p38 activation and ROS elimination. Collectively, our results suggest that ST-L and ST-B exerts potential anti-inflammatory activity by suppressing NF- B and MAPK signaling activation, and activating HO-1 expression through the nuclear accumulation of Nrf2 via ROS-dependent p38 activation. These findings suggest that ST-L and ST-B may have great potential for the development of anti-inflammatory drug to treat acute and chronic inflammatory disorders.

A new nucleoside and two new pyrrole alkaloid derivatives from Cordyceps militaris

A new nucleoside, a new natural product nucleoside, and two new pyrrole alkaloids derivatives with eight known compounds were isolated from the fruiting body of Cordyceps militaris. The structures of the new compounds were elucidated through extensive analysis of spectroscopic data including 1D and 2D NMR, HRESIMS, IR and UV. All the isolated compounds were detected for their bioactivities against LPS-induced NO production in RAW 264.7 cells. Unfortunately, all the isolates have shown no obvious activity.

Terpenoids from the roots of Leontopodium longifolium and their inhibitory activity on NO production in RAW264.7 cells

Phytochemical investigation of the roots of Leontopodium longifolium, led to the isolation of a novel norsesquiterpene, named as longifolactone (1), along with three known diterpenes. The structures of these compounds were elucidated by analysis of HR-ESI-MS, UV, IR and 1D and 2D NMR spectroscopic data. The absolute configuration of the new compound was determined by electronic circular dichroism (ECD) using both experimental and calculated ECD spectra. Furthermore, their anti-inflammatory effects were evaluated in LPS-activated RAW264.7 cells to determine their effects on the release of NO. Longifolactone (1) showed weak cytotoxicity towards two human cancer cell lines.

Synthesis of new ent-labdane diterpene derivatives from andrographolide and evaluation of their anti-inflammatory activities

Two series of andrographolide derivatives with nitrogen-containing heterocycles, phenols and aromatic acids as bioisostere moiety of lactone ring were synthesized. 8 from 18 tested compounds showed stronger inhibitory effect on LPS-induced NO production in RAW264.7 macrophage than hydrocortisone. Among them, compound 8m exhibited the most potent inhibition with IC50 of 3.38 ± 1.03 μM. The structure-activity relationships (SARs) suggested that the replacement of lactone ring with small-molecule phenols could improve the anti-inflammatory efficacy. Furthermore, compound 8m significantly reduced the levels of pro-inflammatory cytokine IL-1β and IL-6 with no influence on cell survival, decreased the expression of iNOS and COX-2, and down-regulated the level and phosphorylation of IκBα, as well as the expression of NF-κB. Also it blocked the nuclear translocation of NF-κB in LPS-induced macrophage. Therefore, the anti-inflammation mechanism of compound 8m was related to the inhibition of COX-2, iNOS and NF-κB signal pathway.

Phenolic constituents from stems of Ilex asprella

Phytochemical investigation on Ilex asprella stems by using various chromatographic techniques led to the isolation of 18 phenolic constituents. Based on spectroscopic data analyses and/or comparison of the spectroscopic data with those in literature, these constituents were identified, including two lignans (1, 2), five phenylpropanes (3-7), six chlorogenic analogues (8-13), and five benzoic analogues (14-18). Among them, compounds 3-7, 9, 11, 13, 14, 17, and 18 were isolated from genus Ilex for the first time, and 2, 8, 10, 15, and 16 were isolated from this species for the first time. The in vitro anti-inflammatory assay results showed that compounds 8, 9, 11, 13, and 15 possessed moderate inhibition on the NO production in RAW264.7 cells with IC₅₀ values of 51.1-85.8 μmol·L⁻¹. The present study brought preliminary reference for the clarification of therapeutic ingredients of I. asprella with anti-inflammatory efficacy and its quality evaluation.