Anti-Inflammatory Effects and Mechanisms of Action of Coussaric and Betulinic Acids Isolated from Diospyros kaki in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Diospyros kaki Thunb. is widely distributed in East Asian countries, its leaves being mainly used for making tea. In this study, coussaric acid (CA) and betulinic acid (BA), both triterpenoid compounds, were obtained from D. kaki leaf extracts through bioassay-guided isolation. CA and BA showed anti-inflammatory effects via inhibition of the nuclear factor-κB (NF-κB) pathway, providing important information on their anti-inflammatory mechanism. Furthermore, they markedly inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and suppressed tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) levels. Furthermore, they decreased protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Pre-treatment with CA and BA inhibited LPS-induced NF-κB. We further examined the effects of CA and BA on heme oxygenase (HO)-1 expression in RAW 264.7 macrophages: BA induced HO-1 protein expression in a dose-dependent manner, while CA had no effect. We also investigated whether BA treatment induced nuclear translocation of Nrf2. BA inhibited LPS-induced NF-κB-binding activity, as well as pro-inflammatory mediator and cytokine production (e.g., NO, PGE2, TNF-α, IL-1β, IL-6), by partial reversal of this effect by SnPP, an inhibitor of HO-1. These findings further elucidate the anti-inflammatory mechanism of CA and BA isolated from D. kaki.


Introduction
Inflammation is an important part of the protective immune response against harmful stimuli. However, uncontrolled inflammation can lead to the development of diseases, such as inflammatory bowel disease, rheumatoid arthritis, neurodegenerative disorders, and sepsis [1]. Lipopolysaccharide (LPS), an exogenous bacterial endotoxin, activates macrophages such that they produce various pro-inflammatory cytokines and mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β

Inhibitory Effects of CA and BA on the Production of Pro-Inflammatory Mediators and Enzymes in LPS-stimulated RAW 264.7 Macrophages
Cytotoxic effects of CA and BA on RAW 264.7 macrophages were determined by using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The viability of cells incubated with different concentrations of CA (10-160 μM) and BA (5-80 μM) was not affected at concentrations up to 80 μM and 10 μM ( Figure 2). Subsequent experiments were conducted at non-toxic concentrations of CA and BA. We evaluated the inhibitory effects of CA and BA on NO and PGE2 production in RAW 264.7 macrophages. RAW 264.7 macrophages were treated with the indicated concentrations of CA and BA for 3 h prior to LPS treatment for 24 h.

Inhibitory Effects of CA and BA on the Production of Pro-Inflammatory Mediators and Enzymes in LPS-stimulated RAW 264.7 Macrophages
Cytotoxic effects of CA and BA on RAW 264.7 macrophages were determined by using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The viability of cells incubated with different concentrations of CA (10-160 μM) and BA (5-80 μM) was not affected at concentrations up to 80 μM and 10 μM ( Figure 2). Subsequent experiments were conducted at non-toxic concentrations of CA and BA. We evaluated the inhibitory effects of CA and BA on NO and PGE2 production in RAW 264.7 macrophages. RAW 264.7 macrophages were treated with the indicated concentrations of CA and BA for 3 h prior to LPS treatment for 24 h. We evaluated the inhibitory effects of CA and BA on NO and PGE 2 production in RAW 264.7 macrophages. RAW 264.7 macrophages were treated with the indicated concentrations of CA and BA for 3 h prior to LPS treatment for 24 h. As shown in Figure 3, CA and BA markedly inhibited the production of NO and PGE 2 in LPS-activated RAW 264.7 macrophages in a dose-dependent manner. To confirm the effect of CA and BA on the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, cells were stimulated with LPS (1 µg/mL) for 24 h in the presence or absence of non-cytotoxic concentrations of CA and BA. As shown in Figure 4, CA and BA suppressed the levels of TNF-α, IL-6, and IL-1β in a dose-dependent manner, as measured by ELISA. As shown in Figure 3, CA and BA markedly inhibited the production of NO and PGE2 in LPS-activated RAW 264.7 macrophages in a dose-dependent manner. To confirm the effect of CA and BA on the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, cells were stimulated with LPS (1 μg/mL) for 24 h in the presence or absence of non-cytotoxic concentrations of CA and BA. As shown in Figure 4, CA and BA suppressed the levels of TNF-α, IL-6, and IL-1β in a dose-dependent manner, as measured by ELISA.   As shown in Figure 3, CA and BA markedly inhibited the production of NO and PGE2 in LPS-activated RAW 264.7 macrophages in a dose-dependent manner. To confirm the effect of CA and BA on the production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, cells were stimulated with LPS (1 μg/mL) for 24 h in the presence or absence of non-cytotoxic concentrations of CA and BA. As shown in Figure 4, CA and BA suppressed the levels of TNF-α, IL-6, and IL-1β in a dose-dependent manner, as measured by ELISA.

Effects of CA and BA on iNOS and COX-2 Expression and NF-κB Activation in LPS-Stimulated RAW 264.7 Macrophages
We investigated the effects of CA and BA on LPS-induced inducible nitric oxide synthase (iNOS) and COX-2 protein upregulation in RAW 264.7 macrophages. Cells were treated with the indicated concentrations of CA and BA for 3 h prior to LPS (1 µg/mL) treatment for 24 h, and the expression of iNOS and COX-2 were measured. As shown in Figure 5, CA and BA decreased the protein expression of iNOS and COX-2, in a dose-dependent manner. We tested to determine whether CA and BA inhibit the phosphorylation and degradation of IκB-α, and the translocation of NF-κB (p65) into the nucleus. As shown in Figure 6, IκB-α was degraded and p65 was translocated after treatment with LPS (30 min) in RAW 264.7 macrophages. However, LPS-induced NF-κB activation was significantly inhibited through pre-treatment with various concentrations of CA and BA for 3 h, in a dose-dependent manner.

Effects of CA and BA on iNOS and COX-2 Expression and NF-κB Activation in LPS-Stimulated RAW 264.7 Macrophages
We investigated the effects of CA and BA on LPS-induced inducible nitric oxide synthase (iNOS) and COX-2 protein upregulation in RAW 264.7 macrophages. Cells were treated with the indicated concentrations of CA and BA for 3 h prior to LPS (1 μg/mL) treatment for 24 h, and the expression of iNOS and COX-2 were measured. As shown in Figure 5, CA and BA decreased the protein expression of iNOS and COX-2, in a dose-dependent manner. We tested to determine whether CA and BA inhibit the phosphorylation and degradation of IκB-α, and the translocation of NF-κB (p65) into the nucleus. As shown in Figure 6, IκB-α was degraded and p65 was translocated after treatment with LPS (30 min) in RAW 264.7 macrophages. However, LPS-induced NF-κB activation was significantly inhibited through pre-treatment with various concentrations of CA and BA for 3 h, in a dose-dependent manner.

Effects of CA and BA on iNOS and COX-2 Expression and NF-κB Activation in LPS-Stimulated RAW 264.7 Macrophages
We investigated the effects of CA and BA on LPS-induced inducible nitric oxide synthase (iNOS) and COX-2 protein upregulation in RAW 264.7 macrophages. Cells were treated with the indicated concentrations of CA and BA for 3 h prior to LPS (1 μg/mL) treatment for 24 h, and the expression of iNOS and COX-2 were measured. As shown in Figure 5, CA and BA decreased the protein expression of iNOS and COX-2, in a dose-dependent manner. We tested to determine whether CA and BA inhibit the phosphorylation and degradation of IκB-α, and the translocation of NF-κB (p65) into the nucleus. As shown in Figure 6, IκB-α was degraded and p65 was translocated after treatment with LPS (30 min) in RAW 264.7 macrophages. However, LPS-induced NF-κB activation was significantly inhibited through pre-treatment with various concentrations of CA and BA for 3 h, in a dose-dependent manner.

Effects of CA and BA on HO-1 Expression and Nrf2 Nuclear Translocation in RAW 264.7 Macrophages
We examined the effects of CA and BA on HO-1 expression in RAW 264.7 macrophages. Various concentrations of BA induced HO-1 protein expression in a dose-dependent manner in cells treated for 12 h ( Figure 7B). In contrast, CA had no effect on the expression of HO-1. Accordingly, we investigated whether BA-induced HO-1 expression is associated with the nuclear translocation of Nrf2. Because Nrf2 plays a crucial role in the transcriptional activation of HO-1 gene expression [29], we specifically investigated whether treatment with BA induces the nuclear translocation of Nrf2. Cells incubated with 10 μM BA for 0.5, 1, and 1.5 h showed increased nuclear Nrf2 levels and decreased cytoplasmic Nrf2 levels ( Figure 8A). In addition, the role of Nrf2 in BA-induced HO-1 expression was studied using a siRNA against Nrf2. RAW 264.7 macrophages were transiently Western blot analysis was performed as described in the Materials and Methods, and representative blots from three independent experiments that showed similar results were chosen. * p < 0.05 as compared with the control group. # p < 0.05 as compared with the group treated with LPS alone.

Effects of CA and BA on HO-1 Expression and Nrf2 Nuclear Translocation in RAW 264.7 Macrophages
We examined the effects of CA and BA on HO-1 expression in RAW 264.7 macrophages. Various concentrations of BA induced HO-1 protein expression in a dose-dependent manner in cells treated for 12 h ( Figure 7B). In contrast, CA had no effect on the expression of HO-1. Accordingly, we investigated whether BA-induced HO-1 expression is associated with the nuclear translocation of Nrf2. Because Nrf2 plays a crucial role in the transcriptional activation of HO-1 gene expression [29], we specifically investigated whether treatment with BA induces the nuclear translocation of Nrf2.

Effects of CA and BA on HO-1 Expression and Nrf2 Nuclear Translocation in RAW 264.7 Macrophages
We examined the effects of CA and BA on HO-1 expression in RAW 264.7 macrophages. Various concentrations of BA induced HO-1 protein expression in a dose-dependent manner in cells treated for 12 h ( Figure 7B). In contrast, CA had no effect on the expression of HO-1. Accordingly, we investigated whether BA-induced HO-1 expression is associated with the nuclear translocation of Nrf2. Because Nrf2 plays a crucial role in the transcriptional activation of HO-1 gene expression [29], we specifically investigated whether treatment with BA induces the nuclear translocation of Nrf2. Cells incubated with 10 μM BA for 0.5, 1, and 1.5 h showed increased nuclear Nrf2 levels and decreased cytoplasmic Nrf2 levels ( Figure 8A). In addition, the role of Nrf2 in BA-induced HO-1 expression was studied using a siRNA against Nrf2. RAW 264.7 macrophages were transiently Cells incubated with 10 µM BA for 0.5, 1, and 1.5 h showed increased nuclear Nrf2 levels and decreased cytoplasmic Nrf2 levels ( Figure 8A). In addition, the role of Nrf2 in BA-induced HO-1 expression was studied using a siRNA against Nrf2. RAW 264.7 macrophages were transiently transfected with Nrf2 siRNA, and then treated with 10 µM BA for 12 h. As shown in Figure

Effects of HO-1 Expression on the Inhibition of Pro-Inflammatory Mediators, Cytokines, and NF-κB Activity by BA in LPS-Stimulated RAW 264.7 Macrophages
To confirm that the anti-inflammatory effect of BA correlated with HO-1 expression via the Nrf2 pathway, we investigated whether the effect of BA-induced HO-1 expression could be reversed by pre-treatment with SnPP, an inhibitor of HO-1. RAW 264.7 macrophages were pre-treated with 10 μM BA for 3 h in the absence or presence of SnPP, followed by LPS stimulation for 24 h. As shown in Figure 9, the inhibitory effects of BA toward LPS-induced NF-κB-binding activity and pro-inflammatory mediator and cytokine production (e.g., NO, PGE2, TNF-α, IL-1β, and IL-6) were partially reversed by SnPP.

Effects of HO-1 Expression on the Inhibition of Pro-Inflammatory Mediators, Cytokines, and NF-κB Activity by BA in LPS-Stimulated RAW 264.7 Macrophages
To confirm that the anti-inflammatory effect of BA correlated with HO-1 expression via the Nrf2 pathway, we investigated whether the effect of BA-induced HO-1 expression could be reversed by pre-treatment with SnPP, an inhibitor of HO-1. RAW 264.7 macrophages were pre-treated with 10 µM BA for 3 h in the absence or presence of SnPP, followed by LPS stimulation for 24 h. As shown in Figure 9, the inhibitory effects of BA toward LPS-induced NF-κB-binding activity and pro-inflammatory mediator and cytokine production (e.g., NO, PGE 2 , TNF-α, IL-1β, and IL-6) were partially reversed by SnPP.

Effects of HO-1 Expression on the Inhibition of Pro-Inflammatory Mediators, Cytokines, and NF-κB Activity by BA in LPS-Stimulated RAW 264.7 Macrophages
To confirm that the anti-inflammatory effect of BA correlated with HO-1 expression via the Nrf2 pathway, we investigated whether the effect of BA-induced HO-1 expression could be reversed by pre-treatment with SnPP, an inhibitor of HO-1. RAW 264.7 macrophages were pre-treated with 10 μM BA for 3 h in the absence or presence of SnPP, followed by LPS stimulation for 24 h. As shown in Figure 9, the inhibitory effects of BA toward LPS-induced NF-κB-binding activity and pro-inflammatory mediator and cytokine production (e.g., NO, PGE2, TNF-α, IL-1β, and IL-6) were partially reversed by SnPP.

Discussion
Medicinal plants have become an essential part of health care, based on increased scientific research [9,10]. Recently, various studies have reported that D. kaki Thunb. (Ebenaceae) has anti-inflammatory [30] and anti-oxidant [31] effects. We tested to isolate CA and BA from fractions of DKLE by NO production, as they have anti-inflammatory properties. CA was obtained as colorless needles with molecular formula C30H46O5, and BA was obtained as a white powder with molecular formula C30H48O3 (Figure 1). The biological activity of CA or BA is still mostly unknown. Therefore, we investigated the anti-inflammatory effects and mechanisms of CA and BA in LPS-stimulated RAW 264.7 macrophages.
Macrophages are critical cells in the development of inflammatory reactions, as they excessively produce or secrete various pro-inflammatory mediators and cytokines [29,30,32]. NO plays an important role in inflammatory response as a pro-inflammatory molecule, which is produced by iNOS. Uncontrolled or excess NO production leads to the development of various inflammatory diseases [33][34][35]. Therefore, inhibition of iNOS and NO expression was assessed for anti-inflammatory potential. We investigated whether BA and CA blocked the production of NO and iNOS protein expression in LPS-stimulated inflammatory condition in RAW 264.7 macrophages ( Figures 3A,C and 5). Cyclooxygenase-2 (COX-2) is involved in the synthesis of PGE2, which produces inflammatory symptoms, including fever and pain [34][35][36]. A number of anti-inflammatory drugs target the suppression of PGE2 production and COX-2 expression. TNF-α, IL-1β, and IL-6 play a key role in triggering and promoting inflammation in macrophages [36]. Therefore, suppression of pro-inflammatory cytokines and mediators is vital to control immune responses. We investigated whether BA and CA, components of D. kaki Thunb. (Ebenaceae), blocked the production of pro-inflammatory cytokines in LPS-induced inflammatory RAW 264.7 macrophages. BA and CA also suppressed the levels of COX-2, and the mRNA level of various pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-12 (Figures 4 and 5). These all findings suggest that BA and CA, at least in LPS-stimulated RAW 264.7 macrophages, exert their anti-inflammatory effects by limiting the expression of pro-inflammatory enzymes and cytokines.
Nuclear factor-κB (NF-κB) is an important transcriptional factor involved in inflammation. Upon activation by external stimuli such as TNF-α and LPS, the IκB protein is phosphorylated and degraded, leading to its translocation into the nucleus [37]. Translocated NF-κB interacts with κB elements in the promoter region of various inflammatory genes, leading to the transcription of pro-inflammatory mediators and cytokines including iNOS, COX-2, NO, PGE2, TNF-α, IL-6, and IL-1β [38,39]. Thus, NF-κB has been regarded as the molecular target in development of therapies for inflammatory diseases [40]. In this study, we examined the inhibitory effects of BA and CA on NF-κB, p50, and p65 translocation, and IκBα phosphorylation and degradation. Following treatment with BA and CA, LPS-induced NF-κB activation and IκBα degradation were inhibited in RAW 264.7 macrophages

Discussion
Medicinal plants have become an essential part of health care, based on increased scientific research [9,10]. Recently, various studies have reported that D. kaki Thunb. (Ebenaceae) has anti-inflammatory [30] and anti-oxidant [31] effects. We tested to isolate CA and BA from fractions of DKLE by NO production, as they have anti-inflammatory properties. CA was obtained as colorless needles with molecular formula C 30 H 46 O 5 , and BA was obtained as a white powder with molecular formula C 30 H 48 O 3 (Figure 1). The biological activity of CA or BA is still mostly unknown. Therefore, we investigated the anti-inflammatory effects and mechanisms of CA and BA in LPS-stimulated RAW 264.7 macrophages.
Macrophages are critical cells in the development of inflammatory reactions, as they excessively produce or secrete various pro-inflammatory mediators and cytokines [29,30,32]. NO plays an important role in inflammatory response as a pro-inflammatory molecule, which is produced by iNOS. Uncontrolled or excess NO production leads to the development of various inflammatory diseases [33][34][35]. Therefore, inhibition of iNOS and NO expression was assessed for anti-inflammatory potential. We investigated whether BA and CA blocked the production of NO and iNOS protein expression in LPS-stimulated inflammatory condition in RAW 264.7 macrophages (Figures 3A,C and 5). Cyclooxygenase-2 (COX-2) is involved in the synthesis of PGE 2 , which produces inflammatory symptoms, including fever and pain [34][35][36]. A number of anti-inflammatory drugs target the suppression of PGE 2 production and COX-2 expression. TNF-α, IL-1β, and IL-6 play a key role in triggering and promoting inflammation in macrophages [36]. Therefore, suppression of pro-inflammatory cytokines and mediators is vital to control immune responses. We investigated whether BA and CA, components of D. kaki Thunb. (Ebenaceae), blocked the production of pro-inflammatory cytokines in LPS-induced inflammatory RAW 264.7 macrophages. BA and CA also suppressed the levels of COX-2, and the mRNA level of various pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-12 (Figures 4 and 5). These all findings suggest that BA and CA, at least in LPS-stimulated RAW 264.7 macrophages, exert their anti-inflammatory effects by limiting the expression of pro-inflammatory enzymes and cytokines.
Nuclear factor-κB (NF-κB) is an important transcriptional factor involved in inflammation. Upon activation by external stimuli such as TNF-α and LPS, the IκB protein is phosphorylated and degraded, leading to its translocation into the nucleus [37]. Translocated NF-κB interacts with κB elements in the promoter region of various inflammatory genes, leading to the transcription of pro-inflammatory mediators and cytokines including iNOS, COX-2, NO, PGE 2 , TNF-α, IL-6, and IL-1β [38,39]. Thus, NF-κB has been regarded as the molecular target in development of therapies for inflammatory diseases [40]. In this study, we examined the inhibitory effects of BA and CA on NF-κB, p50, and p65 translocation, and IκBα phosphorylation and degradation. Following treatment with BA and CA, LPS-induced NF-κB activation and IκBα degradation were inhibited in RAW 264.7 macrophages ( Figure 6). Accordingly, the inhibition of the NF-κB pathway in RAW 264.7 macrophages by BA and CA down-regulated the pro-inflammatory mediators, existing an anti-inflammatory effect.
HO-1 is an inducible rate-limiting enzyme involved in heme catabolism, converting heme to biliverdin, ferrous iron, and carbon monoxide (CO) [41]. Under normal conditions, Nrf2 is complexed with the negative regulator of Nrf2, Kelch-like ECH-associated protein (Keap1) in the cytosol [42]. This complex is disrupted under stressful cellular conditions; Nrf2 separates from Keap1 and translocates into the nucleus, where it binds to the antioxidant response element (ARE), a regulatory element in the promoter regions of phase II enzymes, including HO-1 [43]. In this study, we examined the induction of HO-1 after treatment with BA and CA. HO-1 protein expression increased dose-dependently after treatment with BA, but not CA (Figure 7). In addition, BA also increased the Nrf2 translocation time-dependently ( Figure 8A). Moreover, we investigated HO-1 protein expression after treatment with BA and Nrf2 siRNA. When BA is treated with Nrf2 siRNA simultaneously, HO-1 expression is inhibited ( Figure 8B). As shown in Figure 8B, transient transfection with Nrf2 siRNA completely abolished HO-1 expression by BA, which suggested that BA was associated with HO-1 expression via Nrf2 signaling pathways. Furthermore, the inhibitory effects of BA on the production of inflammatory cytokines in LPS-treated RAW 264.7 macrophages were partially reversed by treatment with SnPP, an inhibitor of HO-1 enzyme activity ( Figure 9). These results suggest that the induction of HO-1 is involved in the inhibitory effects of BA on the production of pro-inflammatory mediators and cytokines via the NF-κB pathway. On the other hand, CA has anti-inflammatory action through only NF-κB pathway, but not HO-1/Nrf2 related pathways.

Cell Culture and Viability Assay
RAW 264.7 macrophages were maintained at a density of 5 × 10 5 cells/mL in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100 units/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM), and were incubated at 37 • C in a humidified atmosphere containing 5% CO 2 . The effect of the various experimental treatments on cell viability was evaluated by determining mitochondrial reductase function with an assay based on the reduction of MTT to formazan crystals. The formation of formazan is proportional to the number of functional mitochondria in the living cells. For the determination of cell viability, 50 µL MTT (2.5 mg/mL) was added to cell suspension (1 × 10 5 cells/mL in each well of the 96-well plates) at a final concentration of 0.5 mg/mL, and the mixture was further incubated for 3-4 h at 37 • C. The formazan formed was dissolved in acidic 2-propanol, and the optical density was measured at 590 nm. The optical density of the formazan formed in the control (untreated) cells was considered as 100% viability.

Determination of Nitrite Production and PGE 2 , TNF-α, IL-1β, and IL-6 Assays
The production of nitrite, a stable end product of NO oxidation, was used as a measure of iNOS activity. The nitrite present in the conditioned medium was determined by using a method based on the Griess reaction. The concentrations of PGE 2 , TNF-α, IL-1β, and IL-6 in the culture medium were determined using ELISA kits (R&D Systems) according to the manufacturer's instructions.

Preparation of Cytosolic and Nuclear Fractions
RAW 264.7 macrophages were homogenized in PER-Mammalian Protein Extraction Buffer (1:20, w/v) (Pierce Biotechnology, Rockford, IL, USA) containing freshly added protease inhibitor cocktail I (EMD Biosciences, San Diego, CA, USA) and 1 mM PMSF. The cytosolic fraction of the cells was prepared by centrifugation at 15,000× g for 10 min at 4 • C. Nuclear and cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology), respectively.

Western Blot Analysis
RAW 264.7 macrophages were harvested and pelleted by using centrifugation at 200× g for 3 min. Then, the cells were washed with phosphate-buffered saline and lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using a Lowry protein assay kit (Sigma Chemical Co.). Thirty micrograms of protein from each sample were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electrophoretically transferred onto a Hybond enhanced chemiluminescence nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk and sequentially incubated with the primary antibody (Santa Cruz Biotechnology and Cell Signaling Technology) and a horseradish peroxidase-conjugated secondary antibody, and then subjected to enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

DNA-Binding Activity of NF-κB
The DNA-binding activity of NF-κB in nuclear extracts was measured using the TransAM kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, 30 µL of complete binding buffer (DTT, herring sperm DNA, and binding buffer AM3) was added to each well. The samples were nuclear extracts from RAW 264.7 macrophages stimulated for 30 min with LPS and treated with different concentrations of compounds. Then, 20 µL of the samples in the complete lysis buffer were added to each well (20 µg of nuclear extract diluted in complete lysis buffer). The plates were incubated for 1 h at room temperature with mild agitation (100 rpm on a rocking platform). After washing each well with wash buffer, 100 µL of diluted NF-κB antibody (1:1000 dilution in 1× antibody-binding buffer) was added to each well, and then the plates were incubated further for 1 h as before. After washing each well with the wash buffer, 100 µL of diluted HRP-conjugated antibody (1:1000 dilution in 1× antibody-binding buffer) was added to each well, followed by 1 h incubation as before. One hundred microliters of developing solution were added to each well for 5 min, followed by the addition of stop solution. Finally, the absorbance of each sample at 450 nm was determined by using a spectrophotometer within 5 min.

Transfection
Cells were transiently transfected with 50 nM of HO-1 siRNA and Nrf2 siRNA for 6 h using Lipofectamine 2000™ (Invitrogen), according to the manufacturer's protocol, and recovered in fresh medium containing 10% fetal bovine serum for 24 h.

Statistical Analysis
Data were expressed as the mean ±SD of at least three independent experiments. To compare three or more groups, one-way analysis of variance followed by the Newman-Keuls post hoc test was used. Statistical analysis was performed by using GraphPad Prism software, version 3.03 (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
In this study, two triterpenoid compounds, CA and BA, obtained from DKLE. BA and CA decreased pro-inflammatory mediators via inhibition of NF-κB pathways in LPS-stimulated RAW 264.7 macrophages. Moreover, only BA induced HO-1 induction via Nrf2 translocation, which was involved in their anti-inflammatory properties. These findings provided information on the mechanism of the anti-inflammatory actions of CA and BA from D. kaki. Additional studies on the biological effects of these compounds are warranted in the future.