1. 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 (IL-1β), interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E
2 (PGE
2) [
2,
3]. Nuclear factor-κB (NF-κB) is a key transcriptional factor involved in immune and inflammatory responses [
4]. In the inactive state, NF-κB exists in the cytoplasm and complexes with the inhibitor of NF-κB (IκB). Heme oxygenase-1 (HO-1) has been recognized as an important molecule in the regulation of inflammation because it inhibits the production of pro-inflammatory cytokines and mediators in activated macrophages [
5,
6,
7]. Nuclear transcription factor-E2-related factor 2 (Nrf2) has been reported to be crucial for HO-1 induction [
8].
Recently, traditional herbal medicines have provided an interesting potential source for new drugs in modern medicine [
9,
10].
Diospyros kaki Thunb. (Ebenaceae) is widely cultivated in East Asian countries, and its leaves are commonly used for making tea. It has been widely used in Asia for the treatment of various diseases, such as atherosclerosis, ischemia, and hypotension [
11,
12,
13,
14,
15]. Previous reports indicate that the beneficial components of
D. kaki include flavonoids, tannins, triterpenoids, and vitamin A [
16]. Triterpenoids in
D. kaki have various biological activities, such as anti-oxidant [
17], anti-diabetic [
18], and anti-tumor [
19] effects. Thus, a detailed study on these triterpenoids would prove significant and valuable for human health. Betulinic acid (BA), a pentacyclic triterpene, has been reported to have various biological activities, such as anti-tumor [
20], anti-inflammatory [
21,
22], and anti-malarial [
23] effects. However, the relationship between BA and HO-1 expression is still unclear. Moreover, the biological activity of coussaric acid (CA) has not yet been elucidated.
In this study, we demonstrated the inhibitory effect of a 70% EtOH extract of D. kaki leaves (DKLE) on the inflammatory reaction in LPS-stimulated RAW 264.7 macrophages. We also obtained CA and BA from DKLE through bioassay-guided isolation, and examined their anti-inflammatory effects.
3. 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
30H
46O
5, and BA was obtained as a white powder with molecular formula C
30H
48O
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 (
Figure 3A,C and
Figure 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 (
Figure 4 and
Figure 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.
4. Materials and Methods
4.1. General Information
NMR spectra were recorded in pyridine by using a JNM ECP-400 spectrometer operating (JEOL, Peabody, MA, USA) at 400 MHz for 1H and at 100 MHz for 13C. Flash column chromatography was performed using octadecyl-functionalized silica gel C18 (12 nm, S-75 μm, YMC, Kyoto, Japan). TLC was carried out on silica gel 60 F254 plates (Merck, Darmstadt, Germany). Dulbecco’s modified Eagle’s medium, fetal bovine serum, and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Small interfering RNA (siRNA) for Nrf2 and antibodies to iNOS, COX-2, phosphor (p)-IκBα, IκBα, p65, PCNA, and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HO-1 and Nrf2 antibodies were obtained from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA). Tin protoporphyrin IX (SnPP), an inhibitor of HO activity, was obtained from Porphyrin Products (Logan, UT, USA). Enzyme-linked immunosorbent assay (ELISA) kits for PGE2, TNF-α, IL-1β, and IL-6 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).
4.2. Sample Preparation
D. kaki Thunb. (Ebenaceae) leaves were obtained from the botanical garden of Wonkwang University, Iksan, Korea, in August 2012. The voucher specimen (WK-2012-08-23) was deposited at the Herbarium of the College of Pharmacy, Wonkwang University (Korea). Dried leaves of D. kaki (114.32 g) were subjected to extraction with 70% EtOH in H2O (3 L) by boiling for 2 h. The 70% EtOH extract (31.31 g) was obtained, and some of the extract (5.13 g) was dissolved in MeOH. Continually, the extract (5.13 g) was subjected to C18-functionalized silica gel open column chromatography and eluted with a stepwise gradient of 20%, 40%, 60%, 80%, and 100% (v/v) of MeOH in H2O (500 mL each). The fraction (101.1 mg) eluted with 80% MeOH was subjected to a silica gel column chromatography (2.7 × 57 cm) by using a gradient elution (CH2Cl2:MeOH = 15:1 to 5:1) to obtain CA (24.1 mg). In addition, the fraction (392.2 mg) eluted with 100% MeOH was subjected to silica gel column chromatography (2.7 × 57 cm) by using a gradient elution (CH2Cl2:EtOAc = 15:1 to 5:1) to obtain BA (13.6 mg). The compounds’ identities were confirmed by TLC and NMR analysis.
4.3. Cell Culture and Viability Assay
RAW 264.7 macrophages were maintained at a density of 5 × 105 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% CO2. 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 × 105 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.
4.4. Determination of Nitrite Production and PGE2, 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 PGE2, 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.
4.5. 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.
4.6. 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).
4.7. 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.
4.8. 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.
4.9. 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).
Kyoung-Su Kim 1,†, 
