Potent anti-inflammatory effects of two quinolinedione compounds,OQ1 and OQ21, mediated by dual inhibition of inducible NO synthase and cyclooxygenase-2

Background and purpose:

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested as key components in various inflammatory diseases. Here we examined the effects of new quinolinedione derivatives, 6-(4-fluorophenyl)-amino-5,8-quinolinedione (OQ1) and 6-(2,3,4-trifluorophenyl)-amino-5,8-quinolinedione (OQ21) on activity and expression of iNOS and COX-2 to explore their anti-inflammatory properties.

Experimental approach:

The effects of OQ1 and OQ21 were assessed on lipopolysaccharide (LPS)-induced iNOS and COX-2 in murine macrophage cell line (RAW264.7), along with isolated enzyme assays to measure enzyme inhibition. Nuclear factor-κB (NFκB) activation pathways were investigated to elucidate mechanisms underlying OQ-mediated suppression of the expression of iNOS and COX-2. In vivo anti-inflammatory activities of OQ compounds were evaluated in mouse ear oedema, induced by topical 12-O-tetradecanoylphorbol-13-acetate (TPA).

Key results:

LPS-induced NO production in RAW264.7 cells was inhibited by OQ1 and OQ21 through the attenuation of iNOS expression as well as iNOS activity. Down-regulation of iNOS followed blocking of NFκB activation, as assessed by inhibitory κB degradation and electrophoretic mobility shift assay for NFκB. Synthesis and accumulation of prostaglandin E₂ were also suppressed by OQ1 and OQ21. LPS-induced COX-2 expression and cellular COX-2 activities were attenuated by OQ1 and OQ21. Consistent with these results, OQ1 showed potent anti-inflammatory effects in mouse ear oedema induced by TPA.

Conclusions and implications:

The novel quinolinedione derivatives, OQ1 and OQ21, showed potent anti-inflammatory activity through dual inhibitory effects on iNOS and COX-2, suggesting that OQ derivatives might provide a new therapeutic modality for chronic inflammatory diseases, refractory to conventional drug therapies.     

Effects of hydroxyl group numbers on the B-ring of 5,7-dihydroxyflavones on the differential inhibition of human CYP 1A and CYP1B1 enzymes

Flavonoids are polyphenols composed of two aromatic rings (A, B) and a heterocyclic ring (C). In order to determine the effects of the number of hydroxyl groups in the B-ring of the flavonoids on human cytochrome P450 (CYP) 1 family enzymes, we evaluated the inhibition of CYP1A-dependent 7-ethoxyresorufin omicron-deethylation activity by chrysin, apigenin and luteolin, using bacterial membranes that co-express human CYP1A1, CYP1A2, or CYP1B1 with human NADPH-cytochrome P450 reductase. Chrysin, which possesses no hydroxyl groups in its B-ring, exhibited the most pronounced inhibitory effects on CYP1A2-dependent EROD activity, followed by apigenin and luteolin. On the contrary, CYP1A1-mediated EROD activity was most potently inhibited by luteolin, which is characterized by two hydroxyl groups in its B-ring, followed by apigenin and chrysin. However, all of the 5,7-dihydroxyflavones were determined to similarly inhibit CYP1B1 activity. Chrysin, apigenin, and luteolin exhibited a mixed-type mode of inhibition with regard to CYP1A2, CYP1B1, and CYP1A1, with apparent Ki values of 2.4, 0.5, and 2.0 microM, respectively. These findings suggested that the number of hydroxyl groups in the B-ring of 5,7-dihydroxyflavone might have some influence on the degree to which CYP1A enzymes were inhibited, but not on the degree to which CYP1B1 enzymes were inhibited.     

Alleviation of hepatic injury by chrysin in cisplatin administered rats: Probable role of oxidative and inflammatory markers

BackgroundCisplatin is an effective and extensively used chemotherapeutic agent to treat range of malignancies, but its therapeutic use is limited because of dose-dependent nephrotoxicity and hepatotoxicity. Several published reports advocate that supplementation with antioxidant can influence cisplatin induced hepatic damage.MethodIn the present study the Wistar rats were subjected to concurrent prophylactic oral treatment of chrysin (25 and 50 mg/kg b.wt.) against the hepatotoxicity induced by intraperitoneal administration of cisplatin (7.5 mg/kg b.wt.). Efficacy of chrysin against the hepatotoxicity was evaluated in terms of biochemical estimation of antioxidant enzyme activities, histopathological changes and expression levels of molecular markers of inflammation.ResultsChrysin ameliorated cisplatin-induced lipid peroxidation, xanthine oxidase activity, glutathione depletion, decrease in antioxidant (catalase, glutathione reductase, superoxide dismutase, glutathione peroxidase and glucose-6 phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-transferase and quinone reductase) enzyme activities. Chrysin also attenuated expression of COX-2, iNOS and levels of NFκB and TNF-α, and hepatic tissue damage which were induced by cisplatin. Histological findings further supported the protective effects of chrysin against cisplatin-induced hepatic damage.ConclusionThe results of the present study demonstrate that oxidative stress and inflammation are closely associated with cisplatin-induced toxicity and chrysin shows the protective efficacy against cisplatin-induced hepatotoxicity possibly via attenuating the oxidative stress and inflammatory response.     

Inducible nitric oxide synthase inhibitors of Chinese herbs III. Rheum palmatum

In this paper, the effects of bioactive compounds of Rheum palmatum L. on the inhibition of NO production from RAW 264.7 cells were explored. Seven main anthraquinone derivatives were isolated from the root of R. palmatum, and of these, emodin and rhein significantly inhibited nitrite production from lipopolysaccharide (LPS)-activated RAW 264.7 cells. The IC(50) values for inhibition of nitrite production by emodin and rhein were 60.7 and 67.3 microM, respectively. After iNOS enzyme activity was stimulated by LPS for 12 h, treatment with emodin or rhein at 20 microg/ml for 18 h did not significantly inhibit NO production. The data show that the inhibitory activity of emodin and rhein is not due to direct inhibition of iNOS enzyme activity. However, expression of iNOS and the COX-2 protein was inhibited by emodin in LPS-activated RAW 264.7 cells, and PGE(2) production was reduced. Rhein also inhibited LPS-induced iNOS protein expression, but not COX-2 or PGE(2) production. On the other hand, inhibition effects on NO production from RAW 264.7 cells were enhanced and cytotoxic effects decreased by co-treatment with emodin and rhein. In conclusion, emodin and rhein are major iNOS inhibitors of R. palmatum and may possibly serve as bioactive substances for anti-inflammation effects.     

Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages

Polygonum cuspidatum water extract (PCWE) was shown to be a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). PCWE was compared to baicalin isolated from Scutellaria baicalensis Georgi and berberine of Coptidis rhizoma and Phellodendri cortex, for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. Both PCWE and the compounds inhibited LPS-induced NO production in a concentration-dependent manner without a cytotoxicity. The decrease in NO production was in parallel with the inhibition of LPS-induced iNOS gene expression by PCWE and the compounds. In contrast, iNOS enzyme activity was not inhibited by PCWE and two agents. In addition, only PCWE inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity, while baicalin or berberine did not. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS protein expression, which was inhibited by these PCWE and two agents, although LPS-induced COX-2 protein expression was not affected by NLA and L-NAME. PCWE inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cell, however, baicalin or berberine did not. From the results, it was concluded that co-treatment with NOS inhibitors and PCWE effectively blocks acute production of NO and inhibits expression of iNOS and COX-2 genes.     

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.     

Effects of hydroxylated flavonoids on the ethoxyresorufin O-deethylase and benzo(α)pyrene hydroxylase

In order to understand the mechanism of action of flavonoids on the drug metabolizing enzyme, cytochrome P4501A1, this study was undertaken to examine the effect of chrysin, morin, myricetin and aminopyrine on the activities of ethoxyresorufin O-deethylase and benzo(α) pyrene hydroxylase in the liver. In the isolated perfused rat liver that was pretreated with 3-methylcholanthrene (3MC), chrysin, morin, myricetin and aminopyrine inhibited the activity of ethoxyresorufin O-deethylase with concentration dependent manner. The isolated liver perfusion with chrysin, morin, myricetin and aminopyrine showed inhibition on the induction of ethoxyresorufin O-deethylase by 3MC. And also, in mouse liver hepa I cells, 3MC-stimulated the benzo(α)pyrene hydroxylase activity which was inhibited by chrysin, morin, myricetin and aminopyrine. These results strongly suggested that hydoxylated flavonoids interfered not only the induction of cytochrome P450IA1 enzymes by 3MC but also the interaction of substrates and enzyme.     

Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21Waf1/Cip1 expression: involvement of p38 mitogen-activated protein kinase

Flavonoids are a broadly distributed class of plant pigments, universally present in plants. They are strong anti-oxidants that can inhibit carcinogenesis in rodents. Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammatory, anti-oxidant properties, promotes cell death, and perturbing cell cycle progression. However, the mechanism by which chrysin inhibits cancer cell growth remains poorly understood. Therefore, we developed an interest in the relationship between MAPK signaling pathways and cell growth inhibition after chrysin treatment in rat C6 glioma cells. Cell viability assay and flow cytometric analysis suggested that chrysin exhibited a dose-dependent and time-dependent ability to block rat C6 glioma cell line cell cycle progression at the G1 phase. Western blotting analysis showed that the levels of Rb phosphorylation in C6 glioma cells exposed to 30 microM chrysin for 24h decreased significantly. We demonstrated the expression of cyclin-dependent kinase inhibitor, p21(Waf1/Cip1), to be significantly increased, but the p53 protein level did not change in chrysin-treated cells. Both cyclin-dependent kinase 2 (CDK2) and 4 (CDK4) kinase activities were reduced by chrysin in a dose-dependent manner. Furthermore, chrysin also inhibited proteasome activity. We further showed that chrysin induced p38-MAPK activation, and using a specific p38-MAPK inhibitor, SB203580, attenuated chrysin-induced p21(Waf1/Cip1) expression. These results suggest that chrysin exerts its growth-inhibitory effects either through activating p38-MAPK leading to the accumulation of p21(Waf1/Cip1) protein or mediating the inhibition of proteasome activity.     

In-vitro and in-vivo anti-inflammatory effect of oxyresveratrol from Morus alba L

The antioxidative effects of mulberroside A and oxyresveratrol obtained from Mori Cortex were examined. Mulberroside A and oxyresveratrol showed an inhibitory effect against FeSO4/H2O2-induced lipid peroxidation in rat microsomes and a scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical. The anti-inflammatory effects of mulberroside A and oxyresveratrol using the carrageenin-induced model of inflammation were investigated in rats. Mulberroside A and oxyresveratrol significantly reduced paw edema. To investigate the mechanism of the anti-inflammatory action of these compounds, we examined the effects of oxyresveratrol on lipopolysaccharide (LPS)-induced responses in murine macrophage cell line RAW 264.7. Exposure of LPS-stimulated cells to oxyresveratrol inhibited nitrite accumulation in the culture medium. Oxyresveratrol also inhibited the LPS-stimulated increase of inducible nitric oxide synthase (iNOS) expression in a concentration-dependent manner; however, it had little effect on iNOS enzyme activity, suggesting that the inhibitory activity of oxyresveratrol is mainly due to the inhibition of iNOS expression rather than iNOS enzyme activity. Oxyresveratrol significantly inhibited LPS-evoked nuclear translocation of NF-kappaB and cyclooxygenase-2 (COX-2) activity in RAW 264.7 cells. The results suggest that the anti-inflammatory properties of oxyresveratrol might be correlated with inhibition of the iNOS expression through down-regulation of NF-kappaB binding activity and significant inhibition of COX-2 activity.     

Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: Comparison with celecoxib

Long standing rheumatoid arthritis (RA) is associated with testicular dysfunction and subfertility. Few studies have addressed the pathogenesis of testicular injury in RA and its modulation by effective agents. Thus, the current study aimed at evaluating the effects of two testosterone boosting agents; chrysin, a natural flavone and celecoxib, a selective COX-2 inhibitor, in testicular impairment in rats with adjuvant arthritis, an experimental model of RA. Chrysin (25 and 50 mg/kg) and celecoxib (5 mg/kg) were orally administered to Wistar rats once daily for 21 days starting 1 h before arthritis induction. Chrysin suppressed paw edema with comparable efficacy to celecoxib. More important, chrysin, dose-dependently and celecoxib attenuated the testicular injury via reversing lowered gonadosomatic index and histopathologic alterations with preservation of spermatogenesis. Both agents upregulated steroidogenic acute regulatory (StAR) mRNA expression and serum testosterone with concomitant restoration of LH and FSH. Furthermore, they suppressed inflammation via abrogation of myeloperoxidase, TNF-α and protein expression of COX-2 and iNOS besides elevation of IL-10. Alleviation of the testicular impairment was accompanied with suppression of oxidative stress via lowering testicular lipid peroxides and nitric oxide. With respect to apoptosis, both agents downregulated FasL mRNA expression and caspase-3 activity in favor of cell survival. For the first time, these findings highlight the protective effects of chrysin and celecoxib against testicular dysfunction in experimental RA which were mediated via boosting testosterone in addition to attenuation of testicular inflammation, oxidative stress and apoptosis. Generally, the 50 mg/kg dose of chrysin exerted comparable protective actions to celecoxib.     

Design and synthesis of new 2,4,5-triarylimidazole derivatives as selective cyclooxygenase (COX-2) inhibitors

A new group of 2,4,5-triarylimidazole derivatives, possessing a methyl sulfonyl pharmacophore, were synthesized and their biological activities were evaluated for cyclooxygenase-2 (COX-2) inhibitory activity. In vitro COX-1/COX-2 structure–activity relationships were determined by varying the substituents at the para position of C-2 phenyl ring. Among the 2,4,5-triarylimidazoles, 2-(4-hydroxy phenyl)-4-(4-methylsulfonylphenyl)-5-phenyl-1H imidazole (11f) was identified as a selective COX-2 inhibitor (COX-2 IC50?=?0.15?μM; selectivity index?=?75) that was less potent than the reference drug celecoxib (COX-2 IC50?=?0.06?μM; SI?=?405). A molecular modeling study where 11f was docked in the binding site of COX-2 showed that the methylsulfonyl pharmacophore group is oriented in the vicinity of the COX-2 secondary pocket (Arg⁵¹³, Phe⁵¹⁸, Gly⁵¹⁹, and Val⁵²³). The structure–activity data acquired indicate that COX-1/COX-2 inhibition is sensitive to the nature of the C-2 phenyl substituents.     

Inhibitory Effects of Epigallocatechin-3-Gallate on Microsomal Cyclooxygenase-1 Activity in Platelets

In this study, we investigated the effect of (–)-epigallocatechin-3-gallate (EGCG), a major component of green tea catechins from green tea leaves, on activities of cyclooxygenase (COX)-1 and thromboxane synthase (TXAS), thromboxane A₂ (TXA₂) production associated microsomal enzymes. EGCG inhibited COX-1 activity to 96.9%, and TXAS activity to 20% in platelet microsomal fraction having cytochrome c reductase (an endoplasmic reticulum marker enzyme) activity and expressing COX-1 (70 kDa) and TXAS (58 kDa) proteins. The inhibitory ratio of COX-1 to TXAS by EGCG was 4.8. These results mean that EGCG has a stronger selectivity in COX-1 inhibition than TXAS inhibition. In special, a nonsteroid anti-inflammatory drug aspirin, a COX-1 inhibitor, inhibited COX-1 activity by 11.3% at the same concentration (50 μM) as EGCG that inhibited COX-1 activity to 96.9% as compared with that of control. This suggests that EGCG has a stronger effect than that of aspirin on inhibition of COX-1 activity. Accordingly, we demonstrate that EGCG might be used as a crucial tool for a strong negative regulator of COX-1/TXA₂ signaling pathway to inhibit thrombotic disease-associated platelet aggregation.     

Chrysin suppresses mast cell-mediated allergic inflammation: involvement of calcium, caspase-1 and nuclear factor-κB

A great number of people are suffering from allergic inflammatory diseases such as asthma, atopic dermatitis, and sinusitis. Therefore discovery of drugs for the treatment of these diseases is an important subject in human health. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid contained in propolis, blue passion flower, and fruits. Several studies reported that chrysin has beneficial effects including anti-tumor and anti-oxidant activities. The aim of the present study was to elucidate whether chrysin modulates the allergic inflammatory reaction and to study its possible mechanisms of action using mast cell-based in vitro and in vivo models. Chrysin inhibited immediate-type systemic hypersensitivity and serum histamine release. Chrysin attenuated immunoglobulin E-mediated local anaphylaxis. These inhibitory effects of chrysin on the systemic and local allergic reaction were more potent than cromolyn, a known anti-allergic drug. Chrysin reduced histamine release from mast cells. The inhibitory effect of chrysin on the histamine release was mediated by the modulation of intracellular calcium. In addition, chrysin decreased gene expression of pro-inflammatory cytokines such as, tumor necrosis factor-α, IL (interleukin)-1β, IL-4, and IL-6 in mast cells. The inhibitory effect of chrysin on the pro-inflammatory cytokine was nuclear factor-κB and caspase-1 dependent. Our findings provide evidence that chrysin inhibits mast cell-derived allergic inflammatory reactions by blocking histamine release and pro-inflammatory cytokine expression, and suggest the mechanisms of action. Furthermore, in vivo and in vitro anti-allergic inflammatory effect of chrysin suggests a possible therapeutic application of this agent in allergic inflammatory diseases.     

Effect of wogonin, a plant flavone from Scutellaria radix, on the suppression of cyclooxygenase-2 and the induction of inducible nitric oxide synthase in lipopolysaccharide-treated RAW 264.7 cells

Plant flavonoids show anti-inflammatory activity both in vitro and in vivo. Some flavonoids, such as flavone derivatives, have been reported previously to inhibit nitric oxide (NO) production by suppressing inducible nitric oxide synthase (iNOS) expression. In this investigation, the effects of wogonin, a potent inhibitor of NO production among the flavonoids tested, on cyclooxygenase-2 (COX-2) induction and activity were elucidated further in connection with iNOS, using a mouse macrophage cell line, RAW 264.7. Wogonin inhibited NO and prostaglandin E(2) (PGE(2)) production from lipopolysaccharide-induced RAW cells with IC(50) values of 31 and 0.3 microM, respectively. When added after the induction of iNOS and COX-2, wogonin inhibited the formation of PGE(2) (IC(50) = 0.8 microM), but not the production of NO. Wogonin inhibited COX-2 activity directly (IC(50) = 46 microM) from the homogenate of aspirin-pretreated RAW cells, as determined by measuring [(14)C]PGE(2) formation from [(14)C]arachidonic acid. However, it did not inhibit iNOS or phospholipase A(2) activity. Western blotting showed that wogonin suppressed the induction of both iNOS and COX-2. Prednisolone also suppressed the induction of iNOS and COX-2. Whereas RU-486 (a steroid receptor antagonist) reversed the suppressive activity of prednisolone, it did not affect the suppressive activity of wogonin, suggesting that the suppressive activity of wogonin is not mediated by binding to a steroid receptor. Results from the present study demonstrated that wogonin is a direct COX-2 inhibitor, as well as an inhibitor of iNOS and COX-2 induction. Wogonin may be a potential agent for use in the treatment of inflammatory diseases.     

Analysis of the mechanism for the anti-inflammatory effect of the anti-rheumatic drug auranofin

Effects of auranofin, an orally active chrysotherapeutic agent, were examined on the production of prostaglandin E2 (PGE2) and nitric oxide (NO) in rat peritoneal macrophages and in RAW 264.7 cells, a murine macrophage-like cell line. Auranofin (1-10 microM) inhibited PGE2 production in rat peritoneal macrophages stimulated with 12-O-tetra-decanoylphorbol 13-acetate (TPA, 16.2 nM) at 8-20 h, but did not affect PGE2 production at 4 h. However, in non-stimulated rat peritoneal macrophages, auranofin increased PGE2 production at 4 h and had no effect on PGE2 production at 8-20 h. It was proved that auranofin (1-10 microM) increased COX (cyclooxygenase)-1-dependent PGE2 production and inhibited COX-2-dependent PGE2 production in rat peritoneal macrophages. Auranofin showed no effect on the enzyme activities of the purified COX-1 and COX-2 proteins. Furthermore, auranofin did not affect the COX-1 protein level, but inhibited the TPA-induced expression of COX-2 protein. Therefore, it was suggested that auranofin inhibited PGE2 production by inhibiting the COX-2 protein induction in TPA-stimulated macrophages. In RAW 264.7 cells, auranofin (0.3-3 microM) inhibited lipopolysaccharide-induced NO synthesis by inhibiting the induction of NO synthase (NOS) protein expression. Auranofin did not affect the enzyme activity of iNOS (inducible NOS). Finally, using rat peritoneal macrophages, the effects of auranofin on PGE2 production and NO production were determined. Auranofin (10 microM) strongly inhibited the production of PGE2 and NO, and the induction of COX-2 protein and NOS protein by TPA. Indomethacin, a COX inhibitor, partially inhibited NO production at the concentration at which PGE2 production was completely inhibited. On the other hand, L-NG-monomethyl-L-arginine acetate (L-NMMA), a NOS inhibitor, partially inhibited PGE2 production. NO production was completely inhibited at the same concentration as shown above. These findings suggest that PGE2 production and NO production partially affect each other. Therefore, the inhibition of PGE2 production by auranofin might be partly due to the inhibition of NO production, and the inhibition of NO production by auranofin be partly due to the inhibition of PGE2 production. In conclusion, auranofin inhibits both PGE2 production and NO production by inhibiting the upregulation of mRNA levels of COX-2 and NOS.     

The structure–activity relationship between oxycoumarin derivatives showing inhibitory effects on iNOS in mouse macrophage RAW264.7 cells

We have investigated the structure–activity relationship between 63 natural oxycoumarin derivatives and their effects on the expression of inducible-nitric oxide synthase (iNOS) induced by lipopolysaccharide. The protein expression of iNOS was screened by Western blot analysis, and four 5,7-dimethoxycoumarins were selected as potent inhibitors of iNOS expression. In terms of structural specificity, the methoxyl group on C-5 and C-7 and the short alkyl chain (1–5 carbons) on C-6 may be essential for the potent activities. These compounds also showed inhibitory effects on nitric oxide generation and mRNA expression of inflammatory mediators, namely, iNOS and COX-2. Interestingly, the inhibitory effect on mRNA expression was specific for iNOS and was not detected for neuronal NOS. It is expected that these compounds will show anti-inflammatory activities via inhibition of the expressions of iNOS and COX-2.