Search Results (30)

Nuclear factor-κB (NF-κB) signaling plays a pivotal role in regulating immune responses and is strongly implicated in cancer progression and inflammation-related diseases. The inhibitory κB kinases (IKKs), particularly IKKα, are central to modulating NF-κB activity, with distinct roles in the canonical and non-canonical signaling pathways. This study investigates the potential of selectively targeting IKKα to develop novel therapeutic strategies. A receptor–ligand interaction pharmacophore model was generated based on the co-crystallized structure of IKKα, incorporating six key features, two hydrogen bond acceptors, two hydrogen bond donors, one hydrophobic region, and one hydrophobic aromatic region. This model was used to virtually screen a diverse natural compound library of 5540 molecules, yielding 82 candidates that matched the essential pharmacophore features. Molecular docking and molecular dynamics simulations were subsequently employed to evaluate binding conformations, stability, and dynamic behavior of the top hits. The end-state free energy calculations (gmx_MMPBSA) further validated the interaction strength and stability of selected compounds. To experimentally confirm their inhibitory potential, key compounds were tested in LPS-stimulated RAW 264.7 cells, where they significantly reduced IκBα phosphorylation. These findings validate the integrative computational-experimental approach and identify promising natural compounds as selective IKKα inhibitors for further therapeutic development in cancer and inflammatory diseases. Full article

This study aimed to assess the anti-inflammatory properties of a bioactive glutamic-alanine rich glycoprotein (GP) derived from Undaria pinnatifida on both LPS-stimulated RAW264.7 cells, peritoneal macrophages, and mouse models of carrageenan- and xylene-induced inflammation, investigating the underlying molecular mechanisms. In both in-vitro and in-vivo settings, GP was found to reduce the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) while also inhibiting the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in response to lipopolysaccharide (LPS) stimulation. GP treatment significantly impeded the nuclear translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway by blocking the phosphorylation of IKKα and IκBα, leading to a reduction in proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Additionally, GP effectively inhibited the activation of mitogen-activated protein kinases (MAPKs), with specific inhibitors of p38 and extra-cellular signal regulated kinase (ERK) enhancing GP’s anti-inflammatory efficacy. Notably, GP administration at 10 mg/kg/day (p.o.) markedly reduced carrageenan-induced paw inflammation and xylene-induced ear edema by preventing the infiltration of inflammatory cells into targeted tissues. GP treatment also downregulated key inflammatory markers, including iNOS, COX-2, IκBα, and NF-κB, by suppressing the phosphorylation of p38 and ERK, thereby improving the inflammatory index in both carrageenan- and xylene-induced mouse models. These findings suggest that marine resources, particularly seaweeds like U. pinnatifida, could serve as valuable sources of natural anti-inflammatory proteins for the effective treatment of inflammation and related conditions. Full article

The inhibitory-kappaB kinases (IKKs) IKKα and IKKβ play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKβ-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKβ in vitro and explain the structure–activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα K_i = 10 nM; IKKβ K_i = 680 nM] and SU1349 [IKKα K_i = 16 nM; IKKβ K_i = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKβ in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents. Full article

Early socialization during lactation is advocated as a feeding strategy to reduce the weaning stress of piglets. However, early socialization has often been accompanied by more frequent aggression between individuals, and its effect on the immune system of piglets has yet to be evaluated. In this study, 89 piglets were raised separately under conventional feeding and early socialization environments. Based on differences in the aggressive behavior of the piglets in different environments during lactation, we further investigated the effects of early socialization on oxidative stress in the spleen of the piglets and the inflammatory responses involved in the canonical nuclear factor kappa-B (NF-κB) signaling pathway. The results revealed that early socialization led to a higher aggression level between individuals (p < 0.01), increased malondialdehyde (MDA) and H₂O₂ levels and inducible nitric oxide synthase (iNOS) activity, and inhibited glutathione (GSH) levels and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) in the piglet spleens (p < 0.05). The mRNA expression levels of the protein kinase A (PKA), inhibitor of kappa B kinase-α (IKK-α), inhibitor of kappa B kinase-β (IKK-β), inhibitor of NF-κB-α (IκB-α), NF-κB(p65), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), iNOS, and heat shock protein (HSP) genes were significantly up-regulated, as well as the protein levels of P-p65, IKK-β, P-IkB-α, pro-IL-1β, and TNF-α. In summary, early socialization caused oxidative stress and inflammatory responses in the spleen of the piglets by inducing ROS production and the activation of the canonical NF-κB pathway. Our study revealed that early socialization significantly increased the ROS level in the piglet spleens and activated the canonical NF-κB signaling pathway, which induced a high expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and COX2) and HSP genes regulated by NF-κB signaling, leading to oxidative stress and the inflammatory response. Full article

Glossogyne tenuifolia Cassini (Hsiang-Ju in Chinese) is a perennial herb native to Taiwan. It was used in traditional Chinese medicine (TCM) as an antipyretic, anti-inflammatory, and hepatoprotective agent. Recent studies have shown that extracts of G. tenuifolia possess various bioactivities, including anti-oxidant, anti-inflammatory, immunomodulation, and anti-cancer properties. However, the pharmacological activities of G. tenuifolia essential oils have not been studied. In this study, we extracted essential oil from air-dried G. tenuifolia plants, then investigated the anti-inflammatory potential of G. tenuifolia essential oil (GTEO) on lipopolysaccharide (LPS)-induced inflammation in murine macrophage cells (RAW 264.7) in vitro. Treatment with GTEO (25, 50, and 100 μg/mL) significantly as well as dose-dependently inhibited LPS-induced pro-inflammatory molecules, such as nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, without causing cytotoxicity. Q-PCR and immunoblotting analysis revealed that the inhibition of NO and PGE₂ was caused by downregulation of their corresponding mediator genes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), respectively. Immunofluorescence and luciferase reporter assays revealed that the inhibition of iNOS and COX-2 genes by GTEO was associated with the suppression of nuclear export and transcriptional activation of the redox-sensitive transcription factor, nuclear factor -κB (NF-κB). In addition, GTEO treatment significantly inhibited phosphorylation and proteosomal degradation of the inhibitor of NF-κB (I-κBα), an endogenous repressor of NF-κB. Moreover, treatment with GTEO significantly blocked the LPS-mediated activation of inhibitory κB kinase α (IKKα), an upstream kinase of the I-κBα. Furthermore, p-cymene, β-myrcene, β-cedrene, cis-β-ocimene, α-pinene, and _D-limonene were represented as major components of GTEO. We found that treatment with p-cymene, α-pinene, and _D-limonene were significantly inhibiting LPS-induced NO production in RAW 264.7 cells. Taken together, these results strongly suggest that GTEO inhibits inflammation through the downregulation of NF-κB-mediated inflammatory genes and pro-inflammatory molecules in macrophage cells. Full article

Household processing of fenugreek seeds and leaves, including soaking, germination, and boiling of the seeds, and air-drying of the leaves, has improved the levels of human consumption of the bitter seeds and increased the shelf life of fresh leaves, respectively. The potential anticancer activity of either unprocessed or processed fenugreek seeds or leaves and the relative expression of pro-apoptotic and anti-apoptotic genes of the studied cancerous cell lines exposed to IC₅₀ crude extracts was investigated to observe the apoptotic-inducing property of this plant as an anticancer agent. The protein expression of IKK-α and IKK-β, as inhibitors of NF-KB which exhibit a critical function in the regulation of genes involved in chronic inflammatory disorders, were studied in the tested cancerous cell lines. In this study, the anticancer activity of household-processed fenugreek leaves and seeds against HepG2, HCT-116, MCF-7, and VERO cell lines was measured using an MTT assay. DNA fragmentation of both HepG2 and MCF-7 was investigated by using gel electrophoresis. RT-PCR was used to evaluate the relative expression of each p53, caspase-3, Bax, and Bcl-2 genes, whereas ELISA assay determined the expression of caspase-3, TNF-α, and 8-OHDG genes. Western blotting analyzed the protein-expressing levels of IKK-α and IKK-β proteins in each studied cell line. Data showed that at 500 µg mL⁻¹, ADFL had the highest cytotoxicity against the HepG2 and HCT-116 cell lines. Although, each UFS and GFS sample had a more inhibitory effect on MCF-7 cells than ADFL. Gel electrophoresis demonstrated that the IC₅₀ of each ADFL, UFS, and GFS sample induced DNA fragmentation in HepG2 and MCF-7, contrary to untreated cell lines. Gene expression using RT-PCR showed that IC₅₀ doses of each sample induced apoptosis through the up-regulation of the p53, caspase-3, and Bax genes and the down-regulation of the Bcl-2 gene in each studied cell line. The relative expression of TNF-α, 8-OHDG, and caspase-3 genes of each HepG2 and MCF-7 cell line using ELISA assays demonstrated that ADFL, UFS, and GFS samples reduced the expression of TNF-α and 8-OHDG genes but increased the expression of the caspase-3 gene. Protein-expressing levels of IKK-α and IKK-β proteins in each studied cell line, determined using Western blotting, indicated that household treatments decreased IKK-α expression compared to the UFS sample. Moreover, the ADFL and SFS samples had the most activity in the IKK-β expression levels. Among all studied samples, air-dried fenugreek leaves and unprocessed and germinated fenugreek seeds had the most anti-proliferative and apoptotic-inducing properties against human HepG2, MCF-7, and HCT-116 cell lines, as compared to the VERO cell line. So, these crude extracts can be used in the future for developing new effective natural drugs for the treatment of hepatocellular, breast, and colon carcinomas. Full article

Age-related macular degeneration (AMD) is an irreversible chronic degenerative pathology that affects the retina. Despite therapeutic advances thanks to the use of anti-vascular endothelial growth factor (VEGF) agents, resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, called Resvega^®, was able to disrupt VEGF-A secretion in human ARPE-19 retina cells. We found that Resvega^® inhibits VEGF-A secretion through decreases in both the PI3K-AKT-mTOR and NFκB signaling pathways. In NFκB signaling pathways, Resvega^® inhibits the phosphorylation of the inhibitor of NFκB, IκB, which can bind NFκB dimers and sequester them in the cytoplasm. Thus, the NFκB subunits cannot migrate to the nucleus where they normally bind and stimulate the transcription of target genes such as VEGF-A. The IκB kinase complex (IKK) is also affected by Resvega^® since the nutraceutical formulation decreases both IKKα and IKKβ subunits and the IKKγ subunit which is required for the stimulation of IKK. Very interestingly, we highlight that Resvega^® could prolong the anti-angiogenic effect of Avastin^®, which is an anti-VEGF agent typically used in clinical practice. Our results suggest that Resvega^® may have potential interest as nutritional supplementation against AMD. Full article

In this study, changes in cell signaling mechanisms in skin cells induced by various wavelengths and intensities of light-emitting diodes (LED) were investigated, focusing on the activity of focal adhesion kinase (FAK) in particular. We examined the effect of LED irradiation on cell survival, the generation of intracellular reactive oxygen species (ROS), and the activity of various cell-signaling proteins. Red LED light increased cell viability at all intensities, whereas strong green and blue LED light reduced cell viability, and this effect was reversed by NAC or DPI treatment. Red LED light caused an increase in ROS formation according to the increase in the intensity of the LED light, and green and blue LED lights led to sharp increases in ROS formation. In the initial reaction to LEDs, red LED light only increased the phosphorylation of FAK and extracellular-signal regulated protein kinase (ERK), whereas green and blue LED lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC, and DPI treatments. Even after 24 h of LED irradiation, the activity of FAK and ERK appeared in cells treated with red LED light but did not appear in cells treated with green and blue LED lights. Furthermore, the activity of caspase-3 was confirmed along with cell detachment. Therefore, our results suggest that red LED light induced mitogenic effects via low levels of ROS–FAK–ERK, while green and blue LED lights induced cytotoxic effects via cellular stress and apoptosis signaling resulting from high levels of ROS. Full article

Cellular senescence is a carefully regulated process of proliferative arrest accompanied by functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation; however, the induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of senescence may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like state in tumor cells. The AKT kinase family, which contains three isoforms that play both unique and redundant roles in cancer progression, is commonly hyperactive in many cancers including melanoma and has been implicated in the regulation of senescence. To interrogate the role of AKT isoforms in Cdk4/6i-induced cellular senescence, we generated isoform-specific AKT knockout human melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. We then evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence, finding that cGAS-STING function was dependent on AKT1, and pharmacologic inhibition of cGAS had little effect on senescence. However, we found SASP factors to require NF-κB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1. In summary, we provide the first evidence of a novel, isoform-specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-κB but independent of cGAS. Full article

FGFC1, an active compound isolated from the culture of marine fungi Stachybotrys longispora FG216, elicits fibrinolytic, anti-oxidative, and anti-inflammatory activity. We have previously reported that FGFC1 inhibited the proliferation, migration, and invasion of the non-small cell lung cancer (NSCLC) cells in vitro. However, the precise mechanisms of FGFC1 on NSCLC and its anti-cancer activity in vivo remains unclear. Hence, this study was focused to investigate the effects and regulatory mechanisms of FGFC1 on two NSCLC cell lines, EGFR-mutant PC9 (ex19del) and EGFR wild-type H1299. Results suggested that FGFC1 significantly inhibited proliferation, colony formation, as well as triggered G0/G1 arrest and apoptosis of PC9 cells in a dose- and time-dependent manner, but no obvious inhibitory effects were observed in H1299 cells. Subsequently, transcriptome analysis revealed that FGFC1 significantly down-regulated 28 genes related to the NF-κB pathway, including IL-6, TNF-α, and ICAM-1 in the PC9 cells. We further confirmed that FGFC1 decreased the expression of protein p-IKKα/β, p-p65, p-IκB, IL-6, and TNF-α. Moreover, NF-κB inhibitor PDTC could strengthen the effects of FGFC1 on the expression of CDK4, Cyclin D1, cleaved-PARP-1, and cleaved-caspase-3 proteins, suggesting that the NF-κB pathway plays a major role in FGFC1-induced cell cycle arrest and apoptosis. Correspondingly, the nuclear translocation of p-p65 was also suppressed by FGFC1 in PC9 cells. Finally, the intraperitoneal injection of FGFC1 remarkably inhibited PC9 xenograft growth and decreased the expression of Ki-67, p-p65, IL-6, and TNF-α in tumors. Our results indicated that FGFC1 exerted anti-cancer activity in PC9 cells via inhibiting the NF-κB signaling pathway, providing a possibility for FGFC1 to be used as a lead compound for the treatment of NSCLC in the future. Full article

In this study, papain-generated casein hydrolysates (CH) with a degree of hydrolysis of 13.7% were subjected to a papain-mediated plastein reaction in the absence or presence of one of the exogenous amino acids—Gly, Pro, and Hyp—to prepare four plastein modifiers, or mixed with one of three amino acids to prepare three mixtures. The assay results confirmed that the reaction reduced free NH₂ for the modifiers and caused amino acid incorporation and peptide condensation. When RAW264.7 macrophages were exposed to the CH, modifiers, and mixtures, these samples promoted macrophage growth and phagocytosis in a dose-dependent manner. In addition, the CH shared similar activity in the cells as the mixtures, while the modifiers (especially the PCH-Hyp prepared with Hyp addition) exerted higher potential than CH, the mixtures, and PCH (the modifier prepared without amino acid addition). The plastein reaction thus enhanced CH bioactivity in the cells. When RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS), the inflammatory cells produced more lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) formation, and caused more four inflammatory mediators (NO, PGE2, TNF-α, and IL-6) and two anti-inflammatory mediators (TGF-β1 and IL-10). However, the PCH-Hyp, PCH, and CH at dose levels of 100 μg/mL could combat against the LPS-induced inflammation. Overall, the PCH-Hyp was more active than the CH and PCH in reducing LDH release, ROS formation, and the secretion of these inflammatory mediators, or in increasing the secretion of the anti-inflammatory mediators. The qPCR and Western blot analysis results further confirmed that these samples had anti-inflammatory effects on the stimulated cells by suppressing the LPS-induced activation of the NF-κB signaling pathway, via regulating the mRNA/miRNA expression of iNOS, IL-6, TNF-α, IL-1β, COX-2, TLR4, IL-10, TGF-β1, miR-181a, miR-30d, miR-155, and miR-148, as well as the protein expression of MyD88, p-IKKα, p-IκBα, p-NF-κB p65, and iNOS, involved in this signaling pathway. In addition, the immunofluorescence assay results revealed that these samples could block the LPS-mediated nuclear translocation of the p65 protein and displayed the same function as the NF-κB inhibitor BAY 11-7082. It was concluded that CH could be endowed with higher anti-inflammatory activity to the macrophages by performing a plastein reaction, particularly that in the presence of exogenous Hyp. Full article

Cisplatin is a platinum-based chemotherapeutic agent for treating solid tumors; however, it presents a risk factor for nephropathy. In the present study, we investigated the antioxidant and anti-inflammatory effects of 3-dehydroxyceanothetric acid 2-methyl ester (3DC2ME) isolated from Ziziphus jujuba Mill. in LLC-PK1 cells following cisplatin-induced cytotoxicity. These cells were exposed to 3DC2ME for 2 h, followed by treatment with cisplatin for 24 h. The treated cells were subjected to cell viability analysis using the Ez-Cytox assay. Reactive oxygen species (ROS) were detected via 2′, 7′- dichlorodihydrofluorescein diacetate (DCFH-DA) staining. In addition, western blotting and fluorescent immunostaining were performed to evaluate protein expressions related to oxidative stress and inflammation pathways. Pretreatment with 3DC2ME protected LLC-PK1 cells from cisplatin-induced cytotoxicity and oxidative stress. In addition, pretreatment with 3DC2ME upregulated heme oxygenase 1 (HO-1) via the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the cisplatin-treated LLC-PK1 cells. Furthermore, the increase in the expressions of IκB kinase α/β (IKKα/β), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in these cells was inhibited. These results provide basic scientific evidence for understanding the antioxidant and anti-inflammatory effects of 3DC2ME isolated from Z. jujuba against cisplatin-induced kidney epithelial cell death. Full article

The fruits of the mulberry tree (Morus alba L.), known as white mulberry, have been consumed in various forms, including tea, beverages, and desserts, worldwide. As part of an ongoing study to discover bioactive compounds from M. alba fruits, the anti-inflammatory effect of compounds from M. alba were evaluated in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Phytochemical analysis of the ethanol extract of the M. alba fruits led to the isolation of 22 compounds. Among the isolated compounds, to the best of our knowledge, compounds 1, 3, 5, 7, 11, 12, and 14–22 were identified from M. alba fruits for the first time in this study. Inhibitory effects of 22 compounds on the production of the nitric oxide (NO) known as a proinflammatory mediator in LPS-stimulated RAW 264.7 macrophages were evaluated using NO assays. Western blot analysis was performed to evaluate the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5). We evaluated whether the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5) following LPS stimulation in RAW 264.7 macrophages occurred because of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB) and activations of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cyclo(L-Pro-L-Val) (5) significantly suppressed phosphorylations of IKKα, IKKβ, IκBα, and NF-κB and activations of iNOS and COX-2 in a concentration-dependent manner. Taken together, these results indicate that cyclo(L-Pro-L-Val) (5) can be considered a potential therapeutic agent for the treatment of inflammation-associated disorders. Full article

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells. Full article

Cornus walteri (Cornaceae), known as Walter’s dogwood, has been used to treat dermatologic inflammation and diarrheal disease in traditional oriental medicine. As part of an ongoing research project to discover natural products with biological activities, the anti-inflammatory potential of compounds from C. walteri in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages were explored. Phytochemical analysis of the methanol extract of the stem and stem bark of C. walteri led to the isolation of 15 chemical constituents. These compounds were evaluated for their inhibitory effects on the production of the proinflammatory mediator nitric oxide (NO) in LPS-stimulated macrophages, as measured by NO assays. The molecular mechanisms underlying the anti-inflammatory activity were investigated using western blotting. Our results demonstrated that among 15 chemical constituents, lupeol and benzyl salicylate inhibited NO production in LPS-activated RAW 264.7 macrophages. Benzyl salicylate was more efficient than N^G-monomethyl-L-arginine mono-acetate salt (L-NMMA) in terms of its inhibitory effect. In addition, the mechanism of action of benzyl salicylate consisted of the inhibition of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), inhibitor of kappa B alpha (IκBα), and nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. Furthermore, benzyl salicylate inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Taken together, these results suggest that benzyl salicylate present in the stem and stem bark of C. walteri has potential anti-inflammatory activity, supporting the potential application of this compound in the treatment of inflammatory diseases. Full article