Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages

This work aimed to elucidate the anti-inflammatory mechanism of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. PL induced heme oxygenase-1 (HO-1) of the RAW264.7 macrophages in concentration- and time-dependent manner. It suppressed induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) through down-regulation of iNOS promoter activity in lipopolysaccharide (LPS)-stimulated macrophages. Zn(II) protoporphyrin IX (ZnPP), a specific inhibitor of HO-1, partly blocked suppression by PL on iNOS promoter activity and NO production, which were elevated in LPS-stimulated macrophages. LPS was able to enhance NO production via reactive oxygen species (ROS) generation, c-Jun NH(2)-terminal kinase (JNK) and c-Jun induction. ZnPP prevented PL from down-regulating ROS generation and JNK activation in LPS-stimulated macrophages. Taken together, PL shows its anti-inflammatory activity via mediation of HO-1 in an in vitro inflammation model.     

Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Aim of the study

This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods

Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results

The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion

These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.     

Suppression of lipopolysaccharide-induced of inducible nitric oxide synthase and cyclooxygenase-2 by Sanguis Draconis, a dragon's blood resin, in RAW 264.7 cells

Sanguis Draconis (SD) is a kind of dragon's blood resin that is obtained from Daemomorops draco (Palmae). It is used in traditional medicine and has shown anti-inflammatory activity in some diseases. In this study, we examined the effects of Sanguis Dranonis ethanol extract (SDEE) on LPS-induced inflammation using RAW 264.7 cells. Our data indicated that SDEE inhibits LPS-stimulated NO, PGE2, IL-1 beta and TNF-alpha release, and iNOS and COX-2 expression. Furthermore, SDEE suppressed the LPS-induced p65 expression of NF-kappa B, which was associated with the inhibition of I kappa B-alpha degradation. We also found that the expression of HO-1 was significantly increased in RAW 264.7 cells by SDEE. These results suggest among possibilities of anti-inflammation that SDEE inhibits the production of NO and PGE2 by the down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB (p65) activation. SDEE can induce HO-1 over-expression in macrophage cells, which indicates that it may possess antioxidant properties. This result means that SEDD its anti-inflammatory effects in macrophages may be through a novel mechanism that involves the action of HO-1. Thus, SD could provide a potential therapeutic approach for inflammation-associated disorders.     

(+)-Nootkatone and (+)-valencene from rhizomes of Cyperus rotundus increase survival rates in septic mice due to heme oxygenase-1 induction

Ethnopharmacological relevance

The rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of inflammatory diseases. However, the mechanism by which extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. The aim of the present study was to test whether heme oxygenase (HO)-1 induction is involved in the anti-inflammatory action of ECR.

Materials and methods

Induction of HO-1 and inhibition of inducible nitric oxide synthase (iNOS)/NO production by ECR and its 12 constituents (3 monoterpenes, 5 sesquiterpenes, and 4 aromatic compounds) were investigated using RAW264.7 cells in vitro. In addition, anti-inflammatory action of ECR and its two active ingredients (nookkatone, valencene) were confirmed in sepsis animal model in vivo.

Results

ECR increased HO-1 expression in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in LPS-activated RAW264.7 cells. Among 12 compounds isolated from ECR, mostly sesquiterpenes induced stronger HO-1 expression than monoterpenes in macrophage cells. Nootkatone and valencene (sesquiterpenes) significantly inhibited iNOS expression and NO production in LPS-simulated RAW264.7 cells. Inhibition of iNOS expression by nootkatone, valencene, and ECR were significantly reduced in siHO-1 RNA transfected cells. Furthermore, all three showed marked inhibition of high mobility group box-1 (HMGB1) in LPS-activated macrophages and increased survival rates in cecal ligation and puncture (CLP)-induced sepsis in mice.

Conclusions

Taken together, we concluded that possible anti-inflammatory mechanism of ECR is, at least, due to HO-1 induction, in which sesquiterpenes such as nootkatone and valencene play a crucial role.     

Effects of bee venom on the pro-inflammatory responses in RAW264.7 macrophage cell line

The purpose of this study is to elucidate the molecular mechanism of anti-inflammatory effect of bee venom (BV), which has been used for the treatment of various inflammatory diseases in oriental medicine. With this aim, we examined the effects of BV on the nitric oxide (NO) production by lipopolysaccharide (LPS) or sodium nitroprusside in RAW264.7 macrophages. We further investigated the effects of BV on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) with RT-PCR in LPS-stimulated RAW264.7 cells. BV suppressed the NO production and decreased the levels of iNOS, COX-2, NF-kappaB and MAPK mRNA in a dose-dependent manner. These results suggest that BV has an anti-inflammatory effect by inhibiting iNOS and COX-2 expression, possibly through suppression of NF-kappaB and MAPK expression.     

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells,indicative of alternative macrophage activation

Ethnopharmacological relevance

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages.

Aim of the study

In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity.

Materials and methods

Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/− LPS.

Results

Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression.

Conclusions

These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.     

Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage

The mushroom Phellinus linteus has been known to exhibit potent biological activity. In contrast to the immuno-potentiating properties of Phellinus linteus, the anti-inflammatory properties of Phellinus linteus have rarely been investigated. Recently, ethanol extract and n-BuOH fractions from Phellinus linteus were deemed most effective in anti-inflammatory activity in RAW 264.7 macrophages. The regulatory mechanisms of Phellinus linteus butanol fractions (PLBF) on the pharmacological and biochemical actions of macrophages involved in inflammation have not been clearly defined yet. In the present study, we tested the role of PLBF on anti-inflammation patterns in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. To investigate the mechanism by which PLBF inhibits NO and PGE2 production as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, we examined the activation of IkappaB and MAPKs in LPS-activated macrophages. PLBF clearly inhibited nuclear translocation of NF-kappaB p65 subunits, which correlated with PLBF's inhibitory effects on IkappaBalpha phosphorylation and degradation. PLBF also suppressed the activation of mitogen-activated protein (MAP) kinases including p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Furthermore, macrophages stimulated with LPS generated ROS via activation of membrane-bound NADPH oxidase, and ROS played an important role in the activation of nuclear factor-kappaB (NF-kappaB) and MAPKs. We demonstrated that PLBF directly blocked intracellular accumulation of reactive oxygen species in RAW 264.7 cells stimulated with LPS much as the NADPH oxidase inhibitors, diphenylene iodonium, and antioxidant pyrrolidine dithiocarbamate did. The suppression of NADPH oxidase also inhibited NO production and iNOS protein expression. Cumulatively, these results suggest that PLBF inhibits the production of NO and PGE2 through the down-regulation of iNOS and COX-2 gene expression via ROS-based NF-kappaB and MAPKs activation. Thus, PLBF may provide a potential therapeutic approach for inflammation-associated disorders.     

Gleditsia sinensis thorns inhibit the production of NO through NF-kappaB suppression in LPS-stimulated macrophages

The thorns of Gleditsia sinensis LAM. (Leguminosae) have been used in traditional medicine for the treatment of inflammatory diseases including swelling, suppuration, carbuncle and skin diseases in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of Gleditsia sinensis thorns in RAW 264.7 macrophages. The aqueous extract of Gleditsia sinensis thorns (AEGS) inhibited LPS-induced NO secretion as well as inducible nitric oxide synthase (iNOS) expression, without affecting cell viability. Furthermore, AEGS suppressed LPS-induced NF-kappaB activation, phosphorylation and degradation of IkappaB-alpha, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). These results suggest that AEGS has the inhibitory effects on LPS-induced NO production and iNOS expression in macrophages through blockade in the phosphorylation of MAPKs, following IkappaB-alpha degradation and NF-kappaB activation.     

The anti-inflammatory effects of Pyrolae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO production

The extract of Pyrolae herba (PH), which has been used as an anti-inflammatory folk remedy in Korea and China, was investigated for its anti-inflammatory action using arachidonic acid, 12-O-tetradecanoylphorbol 13-acetate or carrageenan-induced edema assays. The anti-nociceptive activity of PH was also tested in mice using the acetic acid-induced writhing model. PH showed dose-dependent and significant (P<0.05 at 100-400mg/kg) anti-inflammatory and anti-nociceptive activities in the animal assays. The mechanism of the activities of PH was examined by testing the extract to determine if it inhibits the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) from the murine macrophages, RAW 264.7 cells. Similar to the in vivo activities, both the iNOS expression and NO production were significantly suppressed by PH in a dose-dependent manner. PH also inhibited the activating phosphorylation of p38 MAP kinase and NF-kappaB in these cells. These results provide a scientific basis to explain the effects of PH as an anti-inflammatory folk remedy in Asian countries.     

Molecular mechanism of anti-inflammatory activity of Pluchea indica leaves in macrophages RAW 264.7 and its action in animal models ofinflammation

Ethnopharmacological relevance: Pluchea indica Less.

(Asteraceae) is a Thai medicinal plant used in traditional medicine for the treatment of hemorrhoids, lumbago, leucorrhoea and inflammation. This study investigated the molecular mechanism of anti-inflammatory activity of Pluchea indica leaf extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and also determined its action in acute inflammation animal models.

Materials and methods

The inhibitory effect of Pluchea indica leaf extract on LPS-induced nitric oxide (NO) production was evaluated by Griess reaction. Protein and mRNA expressions were determined by real time RT-PCR and Western blotting analysis, respectively. Inducible nitric oxide synthase (iNOS) promoter activity was evaluated by iNOS promoter based reporter gene assay. In vivo anti-inflammatory effect was examined in ethylphenylpropiolate (EPP)-induced ear edema and carrageenan-induced paw edema in rat models.

Results

Ethyl acetate fraction of ethanol extract of Pluchea indica leaves (EFPI) exhibited the potent inhibitory effect on NO production in LPS-induced macrophages and also inhibited PGE₂ release. EFPI reduced iNOS mRNA and protein expression through suppressed iNOS promoter activity and nuclear translocation of subunit p65 of nuclear factor-κB, but did not inhibit phosphorylation of the mitogen-activated protein kinases (MAPKs). Moreover, EFPI possessed anti-inflammatory activities on acute phase of inflammation as seen in EPP-induced ear edema and carrageenan-induced paw edema inrats.

Conclusions

These data support the pharmacological basis of Pluchea indica plant as a traditional herbal medicine for treatment of inflammation.     

Achyranthes japonica exhibits anti-inflammatory effect via NF-κB suppression and HO-1 induction in macrophages

Ethnopharmacological relevance

The roots of Achyranthes japonica Nakai have been used in traditional herbal medicine for the treatment of edema and arthritis in Korea.

Aim of the study

In this study, we investigated the molecular mechanism responsible for anti-inflammatory effects of the aqueous extract of A. japonica roots (AJ) in LPS-stimulated macrophages.

Materials and methods

Nitric oxide (NO) production and as inducible nitric oxide synthase (iNOS) expression were examined in TG-elicited peritoneal macrophages and RAW 264.7 cells. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by Western blotting and RT-PCR, respectively. The activity of NF-κB and Nrf2 were examined by EMSA, immunocytochemistry or reporter assay.

Results

AJ inhibited LPS-induced NO secretion as well as iNOS expression, without affecting cell viability. Furthermore, AJ suppressed LPS-induced NF-κB activation, degradation of IκB-α, phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. Further study demonstrated that AJ induced heme oxygenase-1 (HO-1) gene expression via nuclear translocation and transactivation of Nrf2. In addition, the inhibitory effects of AJ on iNOS expression were abrogated by small interfering RNA-mediated knock-down of HO-1.

Conclusions

These results suggest that AJ suppresses LPS-induced NO production and iNOS expression in macrophages through the inhibition of IκB/NF-κB and MAPK as well as the Nrf2-mediated HO-1 induction. These findings provide the scientific rationale for anti-inflammatory therapeutic use of A. japonica roots.     

Aloe-emodin from rhubarb (Rheum rhabarbarum) inhibits lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Ethnopharmacological relevance

Rheum rhabarbarum (rhubarb) has long been used for the treatment of inflammation in China and other Asian countries. However, the mechanism underlying the anti-inflammatory activity of this medicinal plant is not fully understood. The present study was designed to investigate the anti-inflammatory effects of anthraquinones, the major constituents in rhubarb, and the molecular mechanism involved in their anti-inflammatory effects.

Materials and methods

RAW264.7 cells were stimulated by lipopolysaccharide (LPS) in the presence or absence of the compounds examined. The proliferation of RAW264.7 cells was assayed by the Alamar-Blue method. The quantity of nitric oxide (NO) was determined by Griess assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. Inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κBα (IκBα), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt/phosphoinositide 3-kinase (PI3K) protein expression levels were determined by Western blotting.

Results

Aloe-emodin markedly suppressed the production of NO, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated RAW264.7 cells with no apparent cytotoxicity. The mRNA expression levels of iNOS, IL-6, and IL-1β genes were also significantly inhibited by aloe-emodin. Western blot analysis showed that aloe-emodin suppressed LPS-induced iNOS protein expression, IκBα degradation, and the phosphorylation of ERK, p38, JNK, and Akt.

Conclusions

These results demonstrate that aloe-emodin is the bioactive component of rhubarb that confers an anti-inflammatory effect through a likely mechanism involving a decrease in pro-inflammatory cytokine production in LPS-induced RAW264.7 macrophages via inhibition of NF-κB, MAPK, and PI3K pathways.     

Pistacia chinensis inhibits NO production and upregulates HO-1 induction via PI-3K/Akt pathway in LPS stimulated macrophage cells

Pistacia chinensis has been used for various purposes in China including as an understock for grafting Pistacia vera. However, little attention was given to its health promoting effects. Therefore, in this study, we investigated the effect of Pistacia chinensis methanolic extract (PCME) containing resorcinol class of phenolic lipids on pro-inflammatory mediators and heme oxygenase-1(HO-1) in lipopolysaccharide stimulated RAW264.7 cells. While PCME (2.5-10 μg/ml) inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interleukin (IL)-6, it up-regulated HO-1 expression. Likewise, PCME inhibited iNOS protein expression, but not COX-2, and reduced nitric oxide (NO) release. Moreover, Phosphorylated c-Jun N-terminal Kinase (JNK) was attenuated dose-dependently in PCME pre-treated RAW264.7 cells. In addition, PCME up-regulated HO-1 protein expression was diminished by pre-treatment of PI-3K inhibitor. Furthermore, nuclear factor erythroid 2 related factor 2 (Nrf2) repressor was attenuated time-dependently during PCME treatment. Taken together, our study showed (for the first time) that PCME inhibited NO production and up-regulated HO-1 induction via PI-3K/Akt pathway, suggesting the role of Pistacia chinensis as potential sources of anti-inflammatory and antioxidant natural compounds.     

Anti-inflammatory effect of ethyl acetate extract from Cissus quadrangularis Linn may be involved with induction of heme oxygenase-1 and suppression of NF-κB activation

Aim of the study

Cissus quadrangularis (family: Vitaceae) has been widely used in traditional herbal medicine for the treatment of hemorrhoids, gastric ulcers and bone healing. In the present study, we determined the anti-inflammatory activity and the molecular mechanism of the ethyl acetate extract of Cissus quadrangularis stem (CQE) in LPS-stimulated RAW 264.7 macrophage cells.

Materials and methods

The inhibitory effect of CQE on LPS-induced nitric oxide (NO) production was evaluated in conditioned media. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by RT-PCR and Western blotting analysis, respectively.

Results

CQE potently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells in a dose-dependent manner. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were suppressed also by CQE as was p65 NF-κB nuclear translocation. Further study demonstrated that CQE by itself induced heme oxygenase-1 (HO-1) gene expression at the protein and mRNA levels in dose- and time-dependent manner. In addition, the inhibitory effects of CQE on NO production were abrogated by a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP).

Conclusions

Collectively, these results suggest that CQE exerts an anti-inflammatory effect in macrophages, at least in part, through the induction of HO-1 expression. These findings provide the scientific rationale for anti-inflammatory therapeutic use of Cissus quadrangularis stem.     

Anti-inflammatory activity of phylligenin, a lignan from the fruits of Forsythia koreana, and its cellular mechanism of action

AIM OF THE STUDY: The fruits of Forsythia koreana have long been used in Chinese medicine to treat inflammatory disorders. However, the pharmacological data is not sufficient to clearly establish a scientific rationale for the anti-inflammatory medicinal use of this plant material, and the search for its active principles has been limited so far. MATERIALS AND METHODS: Phylligenin (lignan) was isolated from the fruits of Forsythia koreana and its anti-inflammatory activity was examined. RESULTS AND CONCLUSION: Phylligenin (1-100 microM) and the methanol extract of Forsythia koreana fruits inhibited cyclooxygenase-2 (COX-2)-mediated prostaglandin E(2) and inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) synthesis from lipopolysaccharide-treated RAW 264.7 cells. In the mechanism study, phylligenin inhibited iNOS expression and nuclear factor-kappaB (NF-kappaB) activation but had no effect on COX-2 expression. Moreover, phylligenin significantly inhibited mouse carrageenan-induced paw edema by intraperitoneal administration (22.1-34.7% inhibition at 12.5-100 mg/kg). These pharmacological properties indicate that phylligenin possesses significant anti-inflammatory activity in vitro and in vivo, and may provide the scientific rationale for anti-inflammatory use of the fruits of Forsythia koreana.     

Sclareol exhibits anti-inflammatory activity in both lipopolysaccharide-stimulated macrophages and the λ-carrageenan-induced paw edema model

Sclareol (1) is a natural fragrance compound used widely in the cosmetic and food industries. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and the λ-carrageenan-induced edema mouse paw model were applied to examine the anti-inflammatory potential of 1 and its possible molecular mechanisms. The experimental results obtained demonstrated that this compound inhibited cell growth, nitric oxide (NO) production, and the expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in LPS-stimulated macrophages. Compound 1 also reduced paw edema, the tissue content of NO, tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), iNOS and COX-2 protein expression, and neutrophil infiltration within the tissues after λ-carrageenan stimulation. The present study suggests that the anti-inflammatory mechanisms of 1 might be related to a decrease of inflammatory cytokines and an increase of antioxidant enzyme activity.     

Inhibitory effects of butanol fraction of the aqueous extract of Forsythia koreana on the nitric oxide production by murine macrophage-like RAW 264.7 cells

The aim of this study was to investigate the effect of butanol fraction of the aqueous extract of Forsythia koreana fruits on the nitric oxide (NO) production and inducible nitric oxide synthesis (iNOS) gene expression in murine macrophage-like RAW 264.7 cells. Butanol fraction alone affected neither NO production nor iNOS gene expression in macrophage-like RAW 264.7 cells. However, the butanol fraction inhibited NO production and iNOS gene expression in RAW 264. 7 cells stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). These findings suggest that inhibition of NO production by this butanol fraction in RAW 264.7 cells stimulated with IFN-gamma plus LPS was due to the suppression of iNOS gene expression.