Article pubs.acs.org/JAFC
Inhibitory Effects of 4‑Hydroxyderricin and Xanthoangelol on Lipopolysaccharide-Induced Inflammatory Responses in RAW264 Macrophages Michiko Yasuda,†,⊥ Kyuichi Kawabata,*,‡ Miki Miyashita,‡ Mayu Okumura,‡ Norio Yamamoto,§ Masakazu Takahashi,‡ Hitoshi Ashida,∥ and Hajime Ohigashi‡ †
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan Department of Bioscience, Faculty of Biotechnology, Fukui Prefectural University, 4-1-1 Matsuoka Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195, Japan § Food Science Research Center, House Wellness Foods Corporation, 3-20 Imoji, Itami, Hyogo 664-0011, Japan ∥ Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan ‡
ABSTRACT: The Japanese herb, Ashitaba (Angelica keiskei Koidzumi), contains two prenylated chalcones, 4-hydroxyderricin and xanthoangelol, which are considered to be the major active compounds of Ashitaba. However, their effects on inflammatory responses are poorly understood. In the present study, we investigated the effects and underlying molecular mechanisms of 4hydroxyderricin and xanthoangelol on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264 mouse macrophages. LPS-mediated production of nitric oxide (NO) was markedly reduced by 4-hydroxyderricin (10 μM) and xanthoangelol (5 μM) compared with their parent compound, chalcone (25 μM). They also inhibited LPS-induced secretion of tumor necrosis factor-alpha (TNF-α) and expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Although chalcone decreased the DNA-binding activity of both activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB), 4-hydroxyderricin and xanthoangelol suppressed only AP-1 and had no effect on NF-κB. On the other hand, all of the tested chalcones reduced the phosphorylation (at serine 536) level of the p65 subunit of NF-κB. 4-Hydroxyderricin and xanthoangelol may be promising for the prevention of inflammatory diseases. KEYWORDS: 4-hydroxyderrin, xanthoangelol, Angelica keiskei Koidzumi (Ashitaba), anti-inflammation, NF-κB, AP-1
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INTRODUCTION Chronic inflammation plays a critical role in the development of several diseases, including asthma, rheumatoid arthritis, atherosclerosis, inflammatory bowel diseases, and various human cancers.1 Infection or injury stimulates inflammatory cells to produce proinflammatory mediators, such as NO, reactive oxygen species, prostaglandins, and cytokines.2 AP-1 and NF-κB are well-known to be an important regulator of the expression of iNOS, COX-2, and TNF-α.3−5 Natural dietary sources, such as fruits, vegetables, herbs, and spices, have been reported to provide beneficial effects for human health,1,2,4 thus drawing attention for their prevention against various diseases. Many types of compounds, including 1′-acetoxychavicol acetate (a phenylpropanoid derived from ginger),6 nobiletin (a citrus polymethoxyflavonoid),6 and xanthohumol (a prenylated chalcone contained in hop),7 exhibit anti-inflammatory activity and decrease the transcriptional activity of AP-1 and NF-κB in inflammatory cells. The Japanese herb, Ashitaba (Angelica keiskei Koidzumi, Apiaceae), is a perennial plant grown along the Pacific coast of Japan (Izu islands and the Izu, Bouso, and Miura peninsulas). Ashitaba is enriched with numerous active compounds such as coumarins, flavanones, and chalcones. Among these compounds, prenylated chalcones 4-hydroxyderricin and xanthoangelol (Figure 1) are more abundant and exhibit anti-bacterial,8 anti-cancer,9−11 and anti-diabetic actions.12,13 However, the © 2013 American Chemical Society
Figure 1. Chemical structure of 4-hydroxyderricin and xanthoangelol.
anti-inflammatory activity of 4-hydroxyderricin and xanthoangelol remains unclear. Although the n-hexane extract of Ashitaba has been found to suppress inflammatory responses in LPS-treated macrophages, active compounds were not identified. 14 Sugii et al. (2005) 15 have reported that xanthoangelol D, a xanthoangelol derivative, suppresses TNFα-induced NF-κB activation in cultured porcine aortic endothelial cells. Also, Ohkura et al. (2011)16 have found that xanthoangelol B and D reduced plasminogen activator Received: Revised: Accepted: Published: 462
September 19, 2013 November 22, 2013 December 26, 2013 December 26, 2013 dx.doi.org/10.1021/jf404175t | J. Agric. Food Chem. 2014, 62, 462−467
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Measurement of TNF-α Production. TNF-α was measured using the L929 fibroblast killing assay with some modifications.19 Briefly, RAW264 macrophages were treated with DMSO, 4hydroxyderricin (10 μM), xanthoangelol (5 μM), chalcone (25 μM), or nobiletin (50 μM) in the presence of LPS as described above. The culture supernatant was collected at 1, 3, 6, 12, and 24 h and diluted (500-fold) with RPMI1640 containing 10% FBS, which were used as the sample for the Killing assay. L929 cells were exposed to 2 μg/mL actinomycin D-containing medium and the sample for 20 h. Viability of L929 cells was measured by the crystal violet staining as described above. TNF-α levels in the supernatants were calculated from a standard curve that was constructed using the viability of L929 cells and the concentration of recombinant mouse TNF-α. Real-Time RT-PCR. RAW264 macrophages were incubated for 30 min in serum-free DMEM containing DMSO or the tested compounds (4-hydroxyderricin (10 μM), xanthoangelol (5 μM), chalcone, (25 μM), nobiletin (50 μM), or 1′-acetoxychavicol acetate (6 μM)). After a further 4 h incubation with LPS, total RNA was isolated from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by cDNA synthesis from 20 ng of total RNA using PrimeScript RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). Thermal cycling was performed on a Thermal Cycler Dice real-time system TP860 (Takara) using SYBR Premix Ex TaqII (Takara). The primers used were purchased from Takara (for primers: β-actin and iNOS) and Rikaken (Nagoya, Japan) (for primer: COX-2). Primer sequences were as follows: β-actin as a control (5′- CATCCGTAAAGACCTCTATGCCAAC-3′ and 5′-ATGGAGCCACCGATCCACA3′), iNOS (5′-TAGGCAGAGATTGGAGGCCTTG-3′ and 5′GGGTTGTTGCTGAACTTCCAGTC-3′), COX-2 (5′-CCCCCACAGTCAAAGACACT-3′ and 5′-AGTTGCTCATCACCCCACTC3′). Western Immunoblot Analysis. RAW264 cells were incubated with DMEM containing DMSO or the tested compounds (4hydroxyderricin (10 μM), xanthoangelol (5 μM), chalcone, (25 μM), nobiletin (50 μM), or 1′-acetoxychavicol acetate (6 μM)) for 30 min and then treated with LPS for a further 2 and 24 h. Whole cell lysates (for iNOS, COX-2, and β-actin, 24 h) and nuclear and cytoplasmic extracts (for p65, lamin B1, and JNK, 2 h) were made with RIPA buffer and the NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce Biotechnology, Rockford, IL, USA), respectively. The cells were lysed with RIPA buffer (50 mM Tris, pH 8.0, 150 mM sodium chloride, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and 1 mM 1,4-dithiothreitol) containing the protease inhibitor cocktail, Complete (Roche, Mannheim, Germany), and the phosphatase inhibitor cocktail, PhosSTOP (Roche). Cell lysates were centrifuged (15000g for 5 min at 4 °C), and the supernatant was used as the whole cell lysate. Nuclear and cytoplasmic extraction was performed according to the manufacturer’s instruction. Protein concentrations were determined via the BioRad protein assay (BioRad Laboratories, Hercules, CA, USA) against the standard curve of γglobulin. Equal amounts (10 μg) of protein were separated by 10% SDS polyacrylamide gel electrophoresis for 60 min at 120 V and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for 60 min at 100 V using the Mini Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories). After blocking with Blocking One or Blocking One-P (Nacalai Tesque, Kyoto, Japan), the membranes were treated with the primary antibodies diluted in Tris buffer (0.1 M Tris, pH 7.5, 0.15 M NaCl, 0.1% (v/v) Tween 20) containing 5% (v/v) Blocking One (for anti-iNOS (1:2000), antiCOX-2 (1:8000), or β-actin (1:500)) or Can Get Signal solution 1 (for total and phospho-p65 (both 1:4000), total and phospho-JNK (both 1:4000), or Lamin B1 (1:2000)) overnight at 4 °C. Corresponding horseradish peroxidase-conjugated secondary antibodies were diluted in Tris buffer (0.1 M Tris, pH 7.5, 0.15 M NaCl, 0.1% (v/v) Tween 20) containing 5% (v/v) Blocking One (for rabbit IgG (1:4000) or goat IgG, (1:2000)) or Can Get Signal solution 2 (for rabbit IgG (1:10000)) and added to the membranes for 1 h at room temperature. The blots were detected by enhanced chemiluminescence using ImmunoStar LD and developed by the LAS4000 mini (Fujifilm, Tokyo, Japan).
inhibitor-1 production in TNF-α-stimulated human umbilical vein endothelial cells. To our knowledge, there have been no reports evaluating an anti-inflammatory action of 4-hydroxyderricin and xanthoangelol. In the current study, we investigated the effects and underlying mechanisms of 4hydroxyderricin, xanthoangelol, and their parent compound chalcone (1,3-diphenyl-prop-2-en-1-one framework)17 on LPSinduced inflammatory responses in the murine macrophage RAW264 cell line.
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MATERIALS AND METHODS
Chemicals. 4-Hydroxyderricin and xanthoangelol were purified as described previously.13 Their purity (>90%) were confirmed by highperformance liquid chromatography. Nobiletin and 1′-acetoxychavicol acetate were kindly donated by Dr. Akira Murakami (Kyoto University, Japan). The anti-iNOS and COX-2 antibodies were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The anti-p65, phospho-p65 (Ser536), c-jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), and lamin B1 antibodies were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG and anti-goat IgG antibodies were obtained from Calbiochem (San Diego, CA, USA) and Sigma (St. Louis, MO, USA), respectively. Anti-β-actin antibody was purchased from Santa Cruz Biotechnology. Can Get Signal immunoreaction enhancer solution was obtained from Toyobo (Osaka, Japan). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) unless specified otherwise. Cell Culture. RAW264 mouse macrophages (RCB0535; RIKEN Bio Resource Center, Tsukuba, Japan) were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma) supplemented with 10% (v/ v) heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL). L929 mouse fibroblast cells (RCB1422, RIKEN Bio Resource Center) were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 containing 10% heat-inactivated FBS, penicillin (100 U/ mL), and streptomycin (100 μg/mL). Cells were grown at 37 °C under a humidified atmosphere of 95% air and 5% CO2. RAW264 cells (2.5 × 105 cells/mL) and L929 cells (2.3 × 105 cells/mL) were seeded (in culture medium) on 96-well plates (for NO and TNF-α measurements), 48-well plates (for western immunoblotting), 12well plates (for real-time reverse transcription-polymerase chain reaction (RT-PCR)), or 60 mm dishes (for electrophoretic mobility shift assay) in the culture medium. After 24 h incubation, cells were used for their respective assay. The final concentration of LPS was 100 ng/mL. Determination of the Production of NO. NO production was determined via the Griess reaction,18 which is based on the amount of nitrite, a stable end-product of NO. Briefly, RAW264 cells were treated with the tested compounds (4-hydroxyderricin (10 μM), xanthoangelol (5 μM), chalcone, (25 μM), nobiletin (50 μM), or 1′acetoxychavicol acetate (6 μM)), which were dissolved in dimethyl sulfoxide (DMSO) in serum-free DMEM for 30 min and then exposed to LPS for a further 24 h. The supernatants (0.1 mL) were mixed with 0.1 mL of Griess reagent 1% (w/v) sulfanilamide and 0.1% (w/v) N(1-naphthyl) ethylenediamine dihydrochloride in 2.5% (v/v) phosphoric acid) in 96-well plates. Absorbance was measured at 543 nm using the SPECTRAmax 190 (Molecular Devices, Sunnyvale, CA, USA). The half-maximal inhibitory concentration (IC50) was defined as the concentration of each compound that inhibited NO production by 50%. Measurement of Cell Viability. Cell viability was measured via crystal violet staining. RAW264 cells were fixed and stained with crystal violet solution (0.2% (w/v) in 2% (v/v) ethanol) for 10 min. The stained cells were then extracted with sodium dodecyl sulfate (SDS) solution (0.5% (w/v) in 50% (v/v) ethanol). The optical density was measured at 570 nm with a reference wavelength of 630 nm using the SPECTRAmax 190. The positive control (DMSO + LPS-treated cells) was taken as 100% cell viability. 463
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inflammatory dietary factors6 and were used as the positive controls, significantly suppressed this response (Figure 2A). Furthermore, 4-hydroxyderricin (5 and 10 μM) and xanthoangelol (2.5−10 μM) significantly inhibited LPS-induced NO production in a concentration-dependent manner, with IC50 values of 5.0 ± 1.2 and 2.9 ± 0.4 μM, respectively. At 10 μM, 4hydroxyderricin and xanthoangelol suppressed the production of NO by 90% and 100%, and both compounds had negligible effects on cell viability at this concentration (Figure 2B). Chalcone (25 μM) significantly reduced NO production. Treatment with each phytochemical alone did not affect the production of NO (data not shown). 4-Hydroxyderricin and Xanthoangelol Suppressed LPS-Induced Production of TNF-α in Mouse Macrophages. We next evaluated the effects of 4-hydroxyderricin and xanthoangelol on the production of TNF-α. LPSstimulated RAW264 cells secreted TNF-α into the culture medium in a time-dependent manner (Figure 3). Xanthoange-
Electrophoretic Mobility Shift Assay. RAW264 macrophages were treated with DMSO or the tested compounds (4-hydroxyderricin (10 μM), xanthoangelol (5 μM), chalcone, (25 μM), nobiletin (50 μM), or 1′-acetoxychavicol acetate (6 μM)) in DMEM for 30 min followed by the exposure to LPS for a further 2 h. Nuclear extracts and protein concentration determination were performed as described above. Equal amounts (7 μg) of protein were subjected to the LightShift chemiluminescent EMSA kit (Thermo Fisher scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The 3′-biotin-labeled oligonucleotide sequences for NF-κB were 5′AGTTGAGGGGACTTTCCCAGGC-3′ and 5′GCCTGGGAAAGTCCCCTCAACT-3′ and for AP-1 were 5′CGCTTGATGACTCACCGGAA-3′ and 5′-TTCCGGCTGAGTCATCAAGCG-3′ (STAR Oligo; Rikaken, Nagoya, Japan). Statistical Analysis. All data are expressed as the mean ± standard deviation from at least three independent experiments. Each treatment for the NO and TNF-α assays was performed in triplicate from at least three independent experiments. Comparisons between groups were analyzed by the Dunnett test. Statistical analysis was performed by SPSS 16.0J (SPSS Inc., Chicago, IL, USA). Significance was reached at values of P < 0.05.
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RESULTS 4-Hydroxyderricin and Xanthoangelol Down-Regulated LPS-Mediated Production of NO in Mouse Macrophages. We first determined the effect of 4-hydroxyderricin and xanthoangelol on LPS-induced NO production (Figure 2A). NO production was induced in RAW264 macrophage cells exposed to LPS for 24 h (Figure 2A), and 6 μM 1′acetoxychavicol acetate or 50 μM nobiletin, which are antiFigure 3. 4-Hydroxyderricin and xanthoangelol inhibit LPS-mediated secretion of TNF-α from RAW264 macrophages. RAW264 cells were treated with 4-hydroxyderricin (4HD, 10 μM), xanthoangelol (XAG, 5 μM), chalcone (CHA, 25 μM), nobiletin (NOB, 50 μM), or DMSO (vehicle, Veh) for 30 min and then stimulated with LPS. The TNF-α level in the culture supernatant at the various time-points was measured using L929 Killing assay as described in Materials and Methods. *p < 0.05.
lol, chalcone, and nobiletin strongly decreased the secretion of TNF-α from 3 to 24 h by 57−94% (P < 0.05), respectively, and 4-hydroxyderricin suppressed it by 40−80% (P < 0.05), respectively (Figure 3). 4-Hydroxyderricin and Xanthoangelol Inhibited LPSInduced Expression of iNOS and COX-2. To assess whether the suppression of NO production by 4-hydroxyderricin or xanthoangelol is due to the reduction in the expression of iNOS, western immunoblots and RT-PCR were performed for iNOS. RAW264 cells treated with LPS for 24 h significantly induced the expression of iNOS protein and mRNA (parts A and B of Figure 4, respectively). All of the tested compounds down-regulated the expression of iNOS protein and mRNA (parts A and B of Figure 4, respectively). Similar responses were found with COX-2 (Figure 4A,B). These data indicate that the prenylated chalcones may attenuate LPS-induced NO production via the suppression of iNOS at the transcriptional level. 4-Hydroxyderricin and Xanthoangelol Reduced the DNA-Binding Activity of AP-1 and Inhibited the Phosphorylation of NF-κB but Not Its DNA-Binding Activity. We then investigated the effects of the prenylated chalcones on transcription factors, AP-1 and NF-κB. 4Hydroxyderricin showed partial inhibition of AP-1 DNAbinding activity, while xanthoangelol and other compounds
Figure 2. 4-Hydroxyderricin and xanthoangelol decrease NO production in LPS-treated RAW264 macrophages. RAW264 cells were incubated with 4-hydroxyderricin (4HD, 1−10 μM), xanthoangelol (XAG, 1−10 μM), chalcone (CHA, 25 μM), nobiletin (NOB, 50 μM), 1′-acetoxychavicol acetate (ACA, 6 μM), or DMSO (vehicle, Veh) for 30 min and then treated with or without LPS (100 ng/mL) for 24 h. The cell culture supernatant was collected and (A) NO production was determined. (B) Quantification of cells stained with crystal violet to estimate the cytotoxicity of the tested phytochemicals. *p < 0.05. 464
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Figure 4. 4-Hydroxyderricin and xanthoangelol reduce the expression levels of iNOS and COX-2 in LPS-treated RAW264 macrophages. RAW264 cells were treated with 4-hydroxyderricin (4HD, 10 μM), xanthoangelol (XAG, 5 μM), chalcone (CHA, 25 μM), nobiletin (NOB, 50 μM), 1′-acetoxychavicol acetate (ACA, 6 μM), or DMSO (vehicle, Veh) for 30 min and then treated with LPS (100 ng/mL) for 24 h for (A) western immunoblots and for 4 h for (B) reversetranscription-polymerase chain reaction. β-Actin and gapdh were used as the internal standard for (A) and (B), respectively. Results shown are representative of three independent experiments. *p < 0.05.
completely abolished it (Figure 5A). A similar pattern of inhibition was observed in the phosphorylation level of JNK (Figure 5A). On the other hand, the prenylated chalcones did not affect the DNA-binding activity of NF-κB (Figure 5B). Interestingly, however, 4-hydroxyderricin and xanthoangelol decreased LPS-induced p65 phosphorylation at Ser536 (Figure 5B). In contrast, chalcone suppressed p65 phosphorylation and reduced its activity (Figure 5B). 1′-Acetoxychavicol acetate decreased the phosphorylation, nuclear translocation, and DNA-binding activity of p65 (Figure 5B). However, nobiletin had no effect on the phosphorylation and the activity of p65 (Figure 5B).
Figure 5. Effects of 4-hydroxyderricin and xanthoangelol on the activation of AP-1 and NF-κB. Nuclear and cytoplasmic extraction, western immunoblotting, and an electrophoretic mobility shift assay were performed on RAW264 cells treated with 4-hydroxyderricin (4HD, 10 μM), xanthoangelol (XAG, 5 μM), chalcone (CHA, 25 μM), nobiletin (NOB, 50 μM), 1′-acetoxychavicol acetate (ACA, 6 μM), or DMSO (vehicle, Veh) for 30 min and followed by LPS (100 ng/mL) for 2 h. The nuclear and cytoplasmic extracts prepared from cells immediately after the preincubation was set as 0 h. (A) JNK phosphorylation and AP-1 DNA-binding activity, (B) p65 phosphorylation and NF-κB-DNA binding activity. The NF-κB complex (indicated by the arrow) was confirmed by a supershift assay using the anti-p65 antibody (data not shown). Lamin B1 was used as the internal standard. Results shown are representative of three independent experiments.
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DISCUSSION Proinflammatory mediators play important roles in chronic inflammation, which is associated with the development of obesity, cancer, cardiovascular diseases, and diabetes mellitus.1,3,4 In the current study, we investigated the antiinflammatory activities of 4-hydroxyderricin and xanthoangelol under in vitro conditions. Our findings reveal that these two compounds significantly decrease the production of NO and TNF-α in LPS-treated RAW264 mouse macrophages. Furthermore, the expression of iNOS and COX-2 genes are reduced. These findings are the first to report that 4hydroxyderricin and xanthoangelol attenuate LPS-mediated inflammatory responses in macrophages. Their parent compound chalcone also display similar effects but at concentrations 5- to 10-fold higher than those of the prenylated chalcones. In addition, xanthoangelol, which has the geranyl group (two isoprene units), showed more strong activity (2fold) than 4-hydroxyderricin, which has the dimethylallyl group (one isoprene unit). This may be because that the prenylation increases hydrophobicity and thus the uptake ratio into the cells. In other types of prenylated chalcones, both xanthohumol
containing the dimethylally group at 3′ position and licochalcone A containing 2-methyl-3-buten-2-yl group at 4 position are known to have an anti-inflammatory activity,7,20 suggesting the position of a prenyl group may be not important for exhibiting an anti-inflammatory activity. 1′-Acetoxychavicol acetate and nobiletin are widely accepted as the potent antiinflammatory and cancer chemopreventive agents.6,21−24 4Hydroxyderricin and xanthoangelol showed the anti-inflammatory activity at a concentration below that of nobiletin and lower toxicity to macrophages compared with 1′-acetoxychavicol acetate because toxic concentrations of the prenylated chalcones and 1′-acetoxychavicol acetate were above 25 and 10 μM, respectively (data not shown). Taken together, 4465
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muscle cells and adipocytes in vitro has been shown to occur via the activation of glucose transporter 4.12,13 Anti-diabetic activities of 4-hydroxyderricin in KK-Ay mice was also reported.12 Nakamura et al. (2012)31 have demonstrated that 4-hydroxyderricin and xanthoangelol are distributed as both aglycone and conjugated forms to various tissues, such as liver, muscle, and fat, when mice are orally administered with Ashitaba extract. Generally, flavonoids are rapidly metabolized via glucuronidation, methoxylation, and/or sulfation and then circulate throughout the body. Interestingly, however, 4hydroxyderricin and xanthoangelol are more abundant as aglycones than glucuronides in plasma and tissues.31 4Hydroxyderricin and xanthoangelol predominantly accumulate in mesenteric fat tissue as aglycones,31 possibly because of the hydrophobicity by the prenyloxy group. Chronic inflammation accompanying hypertrophy of mesenteric fat is known to cause insulin resistance, resulting in abnormal control of blood glucose by skeletal muscle tissue.32 Taken together, 4hydroxyderricin and xanthoangelol may be promising dietary factors for the prevention and improvement of metabolic syndromes. Various components from Ashitaba inhibit inflammatory responses. For example, the coumarin derivative, selinidin, suppresses allergic inflammatory responses in mast cells.33 Xanthoangelol B and D was shown to inhibit TNF-α-induced inflammatory responses in cultured endothelial cells.15,16 Therefore, the Japanese herb, Ashitaba, which is rich in prenylated chalcones, may thus be a promising food source for the prevention of chronic inflammatory diseases In conclusion, our study reveals that 4-hydroxyderricin and xanthoangelol are potent suppressors of inflammatory responses and may have distinct mechanisms of action than the parent compound, chalcone. We will address the investigation of the contribution of prenyl groups to their bioavailability and the elucidation of more details of their action mechanisms in the near future.
hydroxyderricin and xanthoangelol with increased hydrophobicity by the prenyl group may effectively accumulate and work in the cells and hence be more potent than compounds with no prenyl group. The expression of several proinflammatory mediators, including iNOS, COX-2, and TNF-α, are regulated by the transcription factors AP-1 and NF-κB.5 Inflammatory stimuli, such as LPS, activate the pathways of mitogen-activated protein kinase (MAPK) and inhibitor of κB kinase (IKK), which then induce the nuclear translocation and increase the transcriptional activity of AP-1 and NF-κB, respectively. Nobiletin and 1′acetoxychavicol acetate are known to exhibit anti-inflammatory activity via the inhibition of the activation of these transcription factors.6 Like them, 4-hydroxyderricin, xanthoangelol, and chalcone reduced the DNA-binding activity of AP-1 and suppressed the activation of upstream kinase JNK. On the other hand, they reduced the phosphorylation of the p65 subunit of NF-κB, whereas only chalcone completely abolishes the DNAbinding activity of NF-κB. Moreover, all of the tested chalcones do not affect p65 nuclear translocation. In the present study, because the actions of the prenylated chalcones on NF-κB were distinct from those of chalcone, prenylation may have thus affected these mechanisms and bioavailability in macrophages. In the report by Murakami et al. (2005),6 1′-acetoxychavicol acetate inhibited gene transcription of COX-2 via reducing the activation of MAPKs, the translocation of p65, and the transcriptional activity of NF-κB, while nobiletin suppressed the COX-2 expression via reducing NF-κB transcriptional activity without affecting MAPK activation and p65 translocation. They showed nearly the same results in our study (though nobiletin suppressed the phosphorylation of JNK). The inhibition patterns of NF-κB by chalcone and the prenylated chalcones are similar to 1′-acetoxychavicol acetate and nobiletin, respectively. However, other prenylated chalcone xanthohumols have shown the chalcone-like action mechanism, namely the reduction of both p65 phosphorylation at Ser536 and NF-κB DNA-binding acivity.7 Murakami et al. (2005)6 supposed that nobiletin may disrupt binding of NF-κB with transcription coactivators such as p300. Buss et al. (2004)25 have suggested that Ser536 phosphorylation disrupts the hydrogen bond between Ser536 and Asp533, thus leading to a lower and higher affinity of p65 for transcription corepressors and coactivators, respectively. IKKβ is considered to be an essential kinase in LPS-induced p65 phosphorylation at Ser536.26 In addition, LPS-mediated and cAMP-independent activation of the catalytic subunit of protein kinase A increases the transcriptional activity of NF-κB through the phosphorylation of p65 at Ser276.27 Licochalcone A suppressed the expression of inflammatory mediators including TNF-α without affecting nuclear translocation and DNA-binding activity of NFκB.20 This compound inhibited LPS-induced phosphorylation of p65 at Ser276, but not Ser536, resulting in reduced association with coactivator p300.20 Furthermore, the acetylation of NF-κB affects the DNA-binding activity.28,29 Therefore, 4-hydroxyderricin and xanthoangelol may regulate the interaction of NF-κB with transcriptional cofactors by inhibiting phosphorylation at Ser536 and at other sites (e.g., Ser276) and chalcone may suppress the NF-κB activation through the control of phosphorylation and acetylation statuses. 4-Hydroxyderricin and xanthoangelol have been found to suppress adipocyte differentiation by reducing the expression of adipocyte-specific transcription factors.30 Furthermore, enhanced glucose uptake activity by the prenylated chalcones in
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-776-61-6000. Fax: +81-776-61-6015. E-mail:
[email protected]. Present Address ⊥
For M.Y.: Graduate School of Nutritional and Environmental Sciences, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
Funding
This study was partially supported by a Grant-in-Aid for Scientific Research (C) (No. 20580139) and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Dr. Akira Murakami of Kyoto University for a kind donation of nobiletin and 1′-acetoxychavicol acetate. 466
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dx.doi.org/10.1021/jf404175t | J. Agric. Food Chem. 2014, 62, 462−467
