Chalcones from Angelica keiskei Attenuate the ...

JOURNAL OF MEDICINAL FOOD J Med Food 17 (12) 2014, 1306–1313 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.3037

Chalcones from Angelica keiskei Attenuate the Inflammatory Responses by Suppressing Nuclear Translocation of NF-jB Hee Ryun Chang,1,* Hwa Jin Lee,2,* and Jae-Ha Ryu1 1

Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Seoul, Korea. 2 Department of Natural Medicine Resources, Semyung University, Jecheon, Chungbuk, Korea.

ABSTRACT The ethyl acetate-soluble fraction from the ethanolic extract of Angelica keiskei showed potent inhibitory activity against the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated RAW 264.7 cells. We identified seven chalcones (1–7) from EtOAc-soluble fractions through the activity-guided separation. Four active principles, identified as 4hydroxyderrcine (1), xanthoangelol E (2), xanthokeismin A (4), and xanthoangelol B (5), inhibited the production of NO and the expression of proinflammatory cytokines, interleukin (IL)-1b and IL-6, in LPS-activated macrophages. Western blotting and reverse transcription–polymerase chain reaction analysis demonstrated that these chalcones attenuated protein and mRNA levels of inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, these active compounds suppressed the degradation of inhibitory-jBa (I-jBa) and the translocation of nuclear factor jB (NF-jB) into nuclei of LPS-activated macrophages. These data demonstrate that four chalcones (1, 2, 4, and 5) from A. keiskei can suppress the LPSinduced production of NO and the expression of iNOS/COX-2 genes by inhibiting the degradation of I-jBa and nuclear translocation of NF-jB. Taken together, four chalcones from A. keiskei may have efficacy as anti-inflammatory agents.

KEY WORDS: Angelica keiskei anti-inflammatory chalcones inducible nitric oxide synthase nitric oxide

cesses such as septic shock, inflammatory bowel disease, cancer, rheumatoid arthritis, gastritis, and metabolic disorders.10 The macrophage is a very important regulator in inflammatory response and in the production of proinflammatory mediators, including NO.11 Nitric oxide synthase (NOS) catalyzes the oxidative deamination of L-arginine to produce NO. Three isoforms of NOS have been identified: endothelial NOS, neuronal NOS, and iNOS.12 The iNOS can be induced by LPS or cytokines in a variety of immune cells, including macrophages, to produce a large amount of NO as a proinflammatory mediator.13 COX-2 catalyzes the rate-limiting step in the synthesis of prostaglandins (PGs). Two COX isoforms, COX-1 and COX-2, have been identified. The COX-1 is a housekeeping enzyme and is constitutively expressed in most mammalian tissues, whereas COX-2 can be induced by several stimuli and is responsible for the production of large amounts of proinflammatory PGs at the inflammatory site.14 During the course of our search for the natural inhibitors of inflammatory responses, we found that the EtOAc-soluble fraction of A. keiskei exhibited potent inhibitory activity of NO production in LPS-activated macrophage cells. Activityguided isolations were performed, and then seven chalcone compounds were purified as anti-inflammatory principles of A. keiskei. Compounds 1, 2, 4, and 5 among seven chalcones are powerful inhibitors of NO production. Although the effect of xanthoangelol E (2) on the production of inflammatory

INTRODUCTION

A

ngelica keiskei Koidz (Umbelliferae, ‘‘Sinsuncho’’ or ‘‘Tomorrow leaf’’ in Korea) is a perennial herb that is widely cultivated as a green vegetable juice ingredient.1 The fresh leaves of this plant and its dried powder are used for functional food and beverages. It has been reported that A. keiskei contains various bioactive chalcones and coumarins, showing antioxidant,2 antidiabetic,3 chemopreventive,4,5 and anti-inflammatory activities.6,7 Lee et al. reported that the hexane fraction of A. keiskei inhibited the production of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) through downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins and mRNA.8 Another study demonstrated that chalcones isolated from A. keiskei attenuate the production of interleukin-6 (IL-6) in tumor necrosis factor-a (TNF-a)-stimulated osteoblast cells.9 Although inflammation is an important host defense mechanism against tissue injuries and invading pathogens, inflammation also plays a central role in pathological pro*These authors contributed equally to this work. Manuscript received 29 August 2013. Revision accepted 10 August 2014. Address correspondence to: Jae-Ha Ryu, PhD, Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-Gil, Yongsan-Gu, Seoul 140-742, Republic of Korea, E-mail: [email protected]

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mediators in LPS-stimulated macrophages has been investigated,15 little is known about the effect of other chalcone compounds, 4-hydroxyderrcine (1), xanthokeismin A (4), and xanthoangelol B (5), on activated macrophages. In the present study, we investigated the capacity of active compounds (1, 2, 4, and 5) and xanthoangelols and their derivatives to inhibit the production of proinflammatory mediators, cytokines, and the expression of iNOS/COX-2 in LPS-stimulated macrophages. We also examined the possible molecular mechanisms of their activities. MATERIALS AND METHODS Test material The leaves and stems of A. keiskei were collected from Jeju Island, Korea, in January 2008 and authenticated by

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Prof. K.S. Yang at Sookmyung Women’s University. A voucher specimen (No. 0070108) was deposited in the herbarium of Sookmyung Women’s University. The air-dried material (1 kg) was reflux extracted with ethanol (1 L · 3) to yield a crude ethanol extract (300 g), which was successively partitioned with n-hexane, ethyl acetate (EtOAc), and butanol. The EtOAc-soluble fractions (7.8 g) were subjected to silica gel column chromatography. Seven chalcones 1–7 (Fig. 1A) were isolated as active principles from A. keiskei by repeated column chromatography and their purity was analyzed by HPLC (Shimadzu HPLC system with UV monitor at 254 nm; l-Bondapak C18 column, 10 lm, 10 · 300 mm; 60% aqueous MeOH as eluent; flow rate 2.0 mL/min): 4-hydroxyderrcin (1), xanthoangelol E (2), xanthoangelol D (3), xanthokeismin A (4), xanthoangelol B (5), xanthoangelol (6), and xanthoangelol F (7). The structures of these chalcones 1–7 were

FIG. 1. The structures of chalcone compounds from Angelica keiskei (A) and the effects of solvent fractions (B) and chalcone compounds from A. keiskei (C) on LPS-induced NO production. The amount of nitrite in the culture medium was measured by using the Griess reagents. Veh, vehicle; Hx, n-hexane layer; EA, ethyl acetate layer; Bu, butanol layer. The values are expressed as the mean – SD of three individual experiments. *P < .05 indicates significant differences from LPS alone. LPS, lipopolysaccharide; NO, nitric oxide.

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identified by 1H- and 13C-NMR spectra, which were consistent with the previously reported spectroscopic data.2,16,17 Dimethyl sulfoxide (DMSO) was used as a solvent to dissolve the test materials, but final concentrations of DMSO in the assay system were less than 0.1%, which has no effect on cell proliferation or other cellular functions. All test concentrations of active compounds showed no significant effect on cell viability. In the present study, curcumin (20 lM), a representative naturally occurring anti-inflammatory compound, was used as a positive control for the evaluation of anti-inflammatory activities. Cell culture RAW 264.7 cells (a murine macrophage cell line; ATCC, Rockville, MD, USA) were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 lg/mL streptomycin (Life Technologies, Frederick, MD, USA). Cells were incubated at 37C in 5% CO2 in a humidified atmosphere. Measurements of NO in LPS-activated RAW 264.7 cells 5

Cells (1 · 10 cells/mL in 48-well plate) were incubated for 20 h in the absence or presence of test samples with LPS (1 lg/mL). NO was assessed by measuring the accumulated nitrite by the Griess method.18 Briefly, samples (100 lL) of culture media were incubated with 150 lL of the Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine in 2.5% phosphoric acid solution) at room temperature for 10 min in a 96-well microplate. Absorbance at 540 nm was measured by using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The concentration of NO was determined by the sodium nitrite standard curve. Western blot analysis RAW 264.7 cells (5 · 105 cells/60-mm dish) were treated with 1 lg/mL of LPS in the absence or presence of test compounds. Following a 20 h treatment, cells were harvested and gently lysed with a cell lysis buffer (Cell Signaling Technologies, Beverly, MA, USA). Cell lysates were then centrifuged at 10,000 g for 20 min at 4C. Supernatants were collected and protein concentrations were determined by the Bradford method. To prepare cytosol and nuclear extracts, cells were treated with test compounds for 30 min before the activation with 1 lg/mL of LPS. Following a 15 min treatment of LPS, cells were harvested by using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA). Antibodies against iNOS (BD Biosciences, Franklin Lakes, NJ, USA), COX-2 (Cayman Chemical Company, Ann Arbor, MI, USA), I-jBa, and p65 (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA) were used for immunoblot analysis. Reverse transcription–polymerase chain reaction analyses RAW 264.7 cells (5 · 105 cells/60-mm dish) were stimulated for 6 h with LPS (1 lg/mL) in the absence or presence

of test compounds. Total RNA was isolated by TRIzol (Life Technologies) extraction according to the manufacturer’s instructions, and then reverse transcribed into cDNA using reverse transcriptase (Life Technologies) and random hexamer (Cosmo, Seoul, Korea). Then, polymerase chain reaction (PCR) analyses were performed on the aliquots of cDNA preparations to detect the gene expression of iNOS, COX-2, IL-1b, IL-6, and b-actin using a recombinant Taq polymerase (Promega, Madison, WI, USA). Statistical analysis The results are expressed as mean – SD of three experiments, and statistical analysis was performed by one-way analysis of variance and Student’s t-test. A P value of < .05 was considered to indicate a significant difference. RESULTS Effects of chalcones from A. keiskei on the production of NO in LPS-stimulated RAW 264.7 cells The n-hexane, EtOAc, and butanol-soluble fractions from the ethanolic extract of A. keiskei were examined for their ability to inhibit production of LPS-induced NO production in RAW 264.7 macrophage cells. The EtOAc-soluble fraction showed the most potent inhibitory activity on NO production (70% inhibition at 10 lg/mL), whereas LPS treatment dramatically increased the concentrations of NO (Fig. 1B). Based on this result, the activity-guided purification of the EtOAc-soluble fraction was performed and led to the isolation of seven active compounds (1–7). The seven isolated chalcones, 1–7, were examined for inhibitory effects on NO production in LPS-activated macrophages, and IC50 values are shown in Table 1. Since four active compounds 1, 2, 4, and 5 potently suppressed the LPS-induced NO production (IC50 values: < 5 lM), we investigated the inhibitory activities of compounds 1, 2, 4, and 5 under inflammatory conditions. The most potent principle was xanthoangelol E (2) with IC50 values of 2.7 lM for the inhibition of NO production. The dose-dependent reduction of NO production by compounds

Table 1. Inhibitory Effects of Chalcone Compounds Isolated from Angelica keiskei on Nitric Oxide Production in Lipopolysaccharide-Activated RAW 264.7 Cells Compounds 1 2 3 4 5 6 7

Inhibition (%)a at 5 lM

IC50 (lM)b

67 71 11 47 58 20 26

3.1 – 0.2 2.7 – 0.1 > 20 4.7 – 0.4 3.6 – 0.3 > 20 > 20

a Values mean the inhibition (%) of NO production at 5 lM concentration of compounds relative to the LPS control (n = 3). b Fifty percent inhibitory concentration of the compounds on LPS-induced NO production. Values are mean – SD of three experiments. LPS, lipopolysaccharide; NO, nitric oxide.


1, 2, 4, and 5 is shown in Figure 1C. Next, we examined the effects of compounds 1, 2, 4, and 5 on the expression of iNOS, which produces NO as a key mediator of inflammation. Effects of chalcones on the expression of iNOS/COX-2 in LPS-stimulated RAW 264.7 cells To elucidate the mechanism of active compounds for the inhibition of NO production, we examined the effects of the four chalcones on the expression of iNOS protein and mRNA. As shown in Figure 2A, four chalcones, 1, 2, 4, and 5, from A. keiskei attenuated the expression of iNOS protein levels, whereas the protein level of iNOS was markedly upregulated by LPS treatment of RAW 264.7 cells. Moreover, reverse transcription–PCR analysis showed downregulation of iNOS mRNA levels by the treatment with the compounds (Fig. 2B). COX-2 is another key enzyme in the mediation of inflammation by catalyzing the rate-limiting step in PG bio-

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synthesis. In the inflammatory condition, proinflammatory cytokines such as IL-1b and TNF-a induce the expression of COX-2.19,20 Thus, compounds 1, 2, 4, and 5 were investigated if they alter the expressions of COX-2 protein and mRNA in inflammatory conditions. As shown in Figure 2C and D, these compounds suppressed the expression of COX-2 protein and mRNA in LPS-activated macrophages. Furthermore, compound 2 (xanthoangelol E) exerted the greatest decrease in expressions of iNOS/COX-2 protein and mRNA. These results indicate that compounds 1, 2, 4, and 5 affect the expressions of LPS-induced iNOS and COX-2 at the transcriptional level. Effects of chalcones on the mRNA expression of proinflammatory cytokines in LPS-activated RAW 264.7 cells To examine the anti-inflammatory potential of the active four chalcones from A. keiskei, we investigated the effect of

FIG. 2. Effects of chalcones on the expression of LPS-induced iNOS/COX-2 protein and mRNA in RAW 264.7 macrophages. (A, C) Cells were treated with compounds for 20 h during LPS (1 lg/mL) activation. Cell lysates were prepared, and the iNOS, COX-2, and b-actin protein levels were determined by western blotting. (B, D) Cells were treated with compounds for 6 h during LPS (1 lg/mL) activation. The mRNA levels for iNOS, COX-2, and b-actin were determined by RT-PCR from total RNA extracts. The relative intensity of iNOS/COX-2 to b-actin bands was measured by densitometry. The values represent mean – SD of three individual experiments. *P < .05 indicates significant differences from LPS alone. COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; RT-PCR, reverse transcription–polymerase chain reaction.

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compounds 1, 2, 4, and 5 on the LPS-induced mRNA expression of IL-1b and IL-6 in RAW 264.7 cells. Cells were treated with 10 lM of compounds 1, 2, 4, and 5 in the presence of LPS (1 lg/mL) for 6 h. As shown in Figure 3, the IL-6 mRNA levels were downregulated by treatments with compounds 2 and 5. In addition, compound 2 suppressed the expressions of IL-1b mRNA in LPS-stimulated macrophages (Fig. 3). Xanthoangelol E (2) was the strongest suppressor of the proinflammatory cytokines, IL-6 and IL-1b. Effects of chalcones on the I-jBa degradation and nuclear translocation of p65 in LPS-stimulated macrophages Nuclear factor jB (NF-jB) is an important transcription factor complex that controls the expression of cell survival genes as well as proinflammatory enzymes and cytokines such as iNOS, COX-2, IL-1b, and IL-6.21 NF-jB, comprising p50 and p65 subunits, is located in the cytoplasm as an inactive p50/p65 dimer that is physically combined with inhibitor jB (I-jB).22 In response to proinflammatory stimuli, I-jB is phosphorylated, ubiquitinated, and rapidly degraded to release and activate p50/p65. Active NF-jB (p50/p65 dimer) translocates to the nucleus and induces the expression of proinflammatory genes.22 To reveal the molecular mechanism for the suppression of the LPS-induced proinflammatory enzyme and cytokine expressions by the compounds 1, 2, 4, and 5, we tested whether the active compounds affect the LPS-induced I-jBa degradation. The I-jBa was dramatically degraded after a 20 min incubation with LPS (1 lg/mL) and regenerated gradually afterward (Fig. 4A). As shown in Figure 4B, the LPS-induced degradation of I-jBa was suppressed by the treatment of 10 lM of 1, 2, 4, and 5 for 15 min. We also investigated whether compounds prevented the nuclear translocation of the p65 subunit of NF-jB after its release from I-jBa. The nuclear p65 was increased after 10–30 min of treatment with LPS (1 lg/mL) and decreased gradually afterward (Fig. 4A). Treatment with compounds 1, 2, 4, and 5 decreased the level of nuclear p65, as shown in Figure 4B. PARP was used as a loading control of the nuclear extract in the immunoblot experiment. Taken together, these observations indicate that chalcones 1, 2, 4, and 5 decrease the I-jBa degradation and

FIG. 3. Effects of chalcones on the LPS-induced inflammatory cytokines in RAW 264.7 macrophages. Cells were stimulated with LPS in the presence or absence of compounds for 6 h. The levels of IL-1b and IL-6 mRNAs were determined by RT-PCR analysis. b-Actin was used as an internal control. Images are representative of three independent experiments that show similar results. IL, interleukin.

nuclear translocation of NF-jB in LPS-activated macrophages. Curcumin, as a positive control, also inhibited the degradation of I-jBa degradation and nuclear translocation of NF-jB. In addition, it has been reported that curcumin exerts anti-inflammatory properties by suppressing the activation of NF-jB through the inhibition of I-jB kinase (IKK).23 IKK mediates I-jBa degradation by phosphorylation of I-jB and then ubiquitination of p-I-jB.24 In this work, it is not clear whether the chalcones 1, 2, 4, and 5 can inhibit the activity of NF-jB by modulating the activity of IKK, and this should be clarified in further investigations. Mitogen-activated protein kinases (MAPKs) have been involved in the activation of NF-jB and LPS-stimulated iNOS and COX-2 expression.25 Therefore, we assessed the effects of LPS-induced phosphorylation of various MAPKs (p38, JNK, and ERK1/2) by the treatment of compound 2 (xanthoangelol E), which shows the most potent inhibitory activities in LPS-stimulated iNOS and COX-2 expression as well as activation of NF-jB. As shown in Figure 4C, compound 2 suppressed the LPS-induced phosphorylation of p38 and JNK in a dose-dependent manner, but not that of ERK1/2. DISCUSSION A. keiskei has been used as a folk medicine and a health promoting vegetable. A. keiskei contains such nutrients as vitamin A, vitamin K, and dietary fiber in its leaves, stems, and roots.26 It has also been reported that A. keiskei is rich in chalcones, such as 4-hydroxyderricin and xanthoangelol, which are considered to have biological functions.16,27 Chalcones are the starting material for biosynthesis of flavonoids found in usual dietary intake of humans. Chalcones possess a large spectrum of bioactivities such as anti-inflammatory, antifungal, antioxidant, and anticancer activities.28–32 Numerous dietary chalcones and their derivatives have been reported to interfere with inflammatory processes.33–37 In the present study, we isolated seven compounds 1–7 from the extracts of A. keiskei (Fig. 1A) and evaluated their anti-inflammatory properties through activity-guided procedures. The structures were identified as chalcones such as 4-hydroxyderricin (1), xanthoangelol E (2), xanthoangelol D (3), xanthokeismin A (4) xanthoangelol B (5), xanthoangelol (6), and xanthoangelol F (7). We investigated their inhibitory effects on inflammatory responses in LPSactivated RAW 264.7 macrophages. Host tissues and cells are injured by the active macrophages, which release the excessive amounts of various inflammatory mediators, including NO and proinflammatory cytokines, such as IL-1b and IL-6, under inflammatory conditions.11,38 We demonstrated the inhibitory effect of chalcone compounds 1–7 on the production of the proinflammatory mediator, NO (Table 1). Herein, we found that xanthoangelols (2 and 5) as well as 4-hydroxyderricin (1) and xanthokeismin A (4) strongly inhibited NO production, but xanthoangelol D (3), xanthoangelol (6), and xanthoangelol F (7) showed very weak activity. The expression of iNOS and COX-2 (Fig. 2) was


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FIG. 4. Suppression of NF-jB activation by chalcones from A. keiskei and repression of phosphorylation of MAPKs by compound 2 in LPSstimulated macrophages. (A) The I-jBa degradation and the accumulation of p65 in the nucleus by time in LPS-induced RAW 264.7 cells and (B) the effects of chalcones on the degradation of I-jBa and the nuclear translocation of p65 in LPS-activated macrophage cells. Cells were pretreated with compounds for 30 min before LPS treatment for 15 min. Cytoplasmic and nuclear extracts were prepared for the western blotting of I-jBa and p65, respectively. (C) Effect of compound 2 on the activation of MAPKs in LPS-stimulated RAW264.7 cells. Cells were pretreated with compounds for 30 min before LPS stimulation. After treatment with LPS for an additional 15 min, proteins were extracted and the levels of phosphorylated p38, JNK, and ERK1/2 were analyzed by western blotting. Images are representative of three independent experiments that show similar results.

suppressed by the active chalcones 1, 2, 4, and 5 in the LPSactivated inflammatory cell system. Moreover, the levels of IL-6 mRNA were markedly decreased by the active chalcones 2 and 5, and the expressions of IL-1b mRNA were strongly suppressed by the active chalcone 2 in activated RAW 264.7 macrophages (Fig. 3). These data reveal that xanthoangelol E (2) is the most potent anti-inflammatory among the chalcones from A. keiskei, indicating that the side chain with a hydroperoxy group of chalcone is important for augmenting the anti-inflammatory activity. NF-jB is a transcription factor and is a key player in enhancing the expression of proinflammatory genes, including IL-1b and IL-6, as well as a hallmark of inflammation, NO.39 NF-jB is present in the cytoplasm of most cells as a heterodimer comprising p50 and p65, which are bound by the inhibitory protein I-jBa. The various stimuli result in the phosphorylation and degradation of I-jBa, allowing the release of the p50/p65 complex. The released NF-jB translocates to the nucleus and regulates the transcription of target genes by binding to specific DNA sequences.40 We observed that four active chalcones (1, 2, 4, and 5) from A. keiskei decreased the LPS-induced nuclear accumulation of the p65 subunit of NF-jB and inhibited the degradation of I-jBa (Fig. 4B). These results suggest that these active chalcones 1, 2, 4, and 5 stabilize I-jBa and suppress the nuclear translocation of NF-jB.

MAPK signaling has also been implicated in the transcription of inflammatory genes by NF-jB. MAPK family proteins (p38, JNK, and ERK1/2) play major roles in the regulation of inflammatory mediators such as NO and proinflammatory cytokines. The attenuation of either p38, JNK, or ERK1/2 is sufficient to reduce the induction of proinflammatory mediators by LPS.41 To investigate the effect of compound 2 (xanthoangelol E) on the LPS-induced phosphorylation of MAPK family proteins, we assessed the levels of phosphorylated p38, JNK, and ERK1/2 in LPSactivated RAW 264.7 cells. The results demonstrated that the phosphorylation of p38, JNK, and ERK1/2 was increased by LPS, while the phosphorylation of p38 and JNK was suppressed by the treatment of xanthoangelol E (2). This observation suggests that the prevention of LPSinduced NF-jB activation by xanthoangelol E (2) might be associated with p38 and JNK inhibition. Taken together, four active chalcones, 1, 2, 4, and 5, from A. keiskei significantly suppressed the production of the inflammatory mediator such as NO and the expressions of iNOS and COX-2 mRNA and protein by inhibiting I-jBa degradation and suppression of the nuclear translocation of NF-jB in LPS-activated macrophages. These active chalcones, 4-hydroxyderricin (1), xanthoangelol E (2), xanthokeismin A (4), and xanthoangelol B (5), from A. keiskei may be beneficial for the treatment of inflammatory diseases.

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ACKNOWLEDGMENT This work was supported by the Sookmyung Women’s University Research Grant in 2011. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Kim OK, Kung SS, Park WB, et al.: The nutritional components of the roots of Angelica keiskei Koidz. Korean J Food Sci Technol 1992;24:592–596. 2. Aoki N, Muko M, Ohta E, et al.: C-Geranlyated chalcones from the stems of Angelica keiskei with superoxide-scavenging activity. J Nat Prod 2008;71:1308–1310. 3. Ohnogi H, Hayami S, Kudo Y, et al.: Angelica keiskei extract improves insulin resistance and hypertriglyceridemia in rats fed a high-fructose drink. Biosci Biotechnol Biochem 2012;76:928– 932 4. Tabata K, Motani K, Takayanagi N, et al.: Xanthoangelol, a major chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma and leukemia cells. Biol Pharm Bull 2005;28: 1404–1407. 5. Akihisa T, Kikuchi T, Nagai H, et al.: 4-Hydroxyderricin from Angelica keiskei roots induces caspase-dependent apoptotic cell death in HL60 human leukemia cells. J Oleo Sci 2011;60:71–77. 6. Ohkura N, Nakakuki Y, Taniguchi M, et al.: Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production. Biofactors 2011;37:455–461. 7. Shin HJ, Shon DH, Youn HS: Isobavachalcone suppresses expression of inducible nitric oxide synthase induced by Toll-like receptor agonists. Int Immunopharmacol 2013;15:38–41. 8. Lee HJ, Choi TW, Kim HJ, et al.: Anti-inflammatory activity of Angelica keiskei through suppression of mitogen-activated protein kinases and nuclear factor-kappaB activation pathways. J Med Food 2010;13:691–699. 9. Shin JE, Choi EJ, Jin Q, et al.: Chalcones isolated from Angelica keiskei and their inhibition of IL-6 production in TNF-a-stimulated MG-63 cell. Arch Pharm Res 2011;34:437–442. 10. Kundu JK, Surh YJ: Inflammation: gearing the journey to cancer. Mutat Res 2008;659:15–30. 11. Lin WW, Karin M: A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest 2007;117: 1175–1183 12. Lowenstein CJ, Hill SL, Lafond-Walker A, et al.: Nitric oxide inhibits viral replication in murine myocarditis. J Clin Invest 1996;97:1837–1843. 13. Aktan F: iNOS-mediated nitric oxide production and its regulation. Life Sci 2004;75:639–653. 14. Lee SH, Soyoola E, Chanmugam P, et al.: Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem 1992;267:25934–25938. 15. Seoa JH, Kim SJ: The anti-inflammatory mechanism of Xanthoangelol E is through the suppression of NF-jB/caspase-1 activation in LPS-stimulated mouse peritoneal macrophage. J Exp Biomed Sci 2012;18:345–354. 16. Baba K, Nakata K, Taniguchi M, et al.: Chalcone from Angelica keiskei. Phytochemistry 1990;29:3907–3910.

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