Kudzu Leaf Extract Suppresses the Production Of

International Journal of Molecular Sciences

Article Kudzu Leaf Extract Suppresses the Production of Inducible Nitric Oxide Synthase, Cyclooxygenase-2, Tumor Necrosis Factor-Alpha, and Interleukin-6 via Inhibition of JNK, TBK1 and STAT1 in Inﬂammatory Macrophages

Seok Hyun Eom 1, So-Jung Jin 1, Hee-Yeong Jeong 2, Youngju Song 3, You Jin Lim 1, Jong-In Kim 4, Youn-Hyung Lee 1 and Hee Kang 2,* ID

1 Department of Horticultural Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 17104, Korea; [email protected] (S.-H.E.); [email protected] (S.-J.J.); [email protected] (Y.-J.L.); [email protected] (Y.-H.L.) 2 Graduate School of East-West Medical Science, Kyung Hee University, Yongin 17104, Korea; [email protected] 3 Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, Seoul 02447, Korea; [email protected] 4 Division of Acupuncture and Moxibustion Medicine, Kyung Hee Korean Medicine Hospital, Kyung Hee University, Seoul 02447, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-31-201-3854

Received: 17 April 2018; Accepted: 18 May 2018; Published: 22 May 2018

Abstract: Kudzu (Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep) is a perennial leguminous vine, and its root and flower have been used for herbal medicine in Asia for a long time. Most dietary flavonoids are reported to be concentrated in its root, not in its aerial parts including leaves. In this study, we investigated whether kudzu leaf and its major constituent, robinin (kaempferol-3-O-robinoside-7-O-rhanmoside) possessed anti-inflammatory activity. To test this hypothesis, we used peritoneal macrophages isolated from BALB/c mice and stimulated the cells with lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. Compared with kudzu root extract, its leaf extract was more potent in inhibiting the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-α, and interleukin-6. Kudzu leaf extract decreased LPS-induced activation of c-Jun N-terminal kinase (JNK) and TANK-binding kinase 1(TBK1) with no effects on nuclear factor-κB and activator protein 1 transcriptional activity. Also, kudzu leaf extract inhibited LPS/IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) activation partly via an altered level of STAT1 expression. Robinin, being present in 0.46% of dry weight of leaf extract, but almost undetected in the root, decreased iNOS protein involving modulation of JNK and STAT1 activation. However, robinin showed no impact on other inflammatory markers. Our data provide evidence that kudzu leaf is an excellent food source of as yet unknown anti-inflammatory constituents.

Keywords: kudzu leaf; robinin; inﬂammation; macrophages; inducible nitric oxide synthase; STAT1

1. Introduction Inﬂammation is a protective response that eliminates noxious stimuli and promotes the regeneration of damaged tissue. Generally, this response begins when phagocytes detect harmful substances from microbes or dying cells through pattern recognition receptors such as toll-like receptors

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(TLRs) [1]. Several peptide and lipid mediators are synthesized by phagocytes and other immune cells nearby, resulting in vasodilation, increased capillary permeability, and vascular stickiness [1]. This brings white blood cells out of the blood vessels to migrate to the site and participate in the ongoing inflammatory response in cooperation with the resident phagocytes. Because excessive production of inflammatory mediators can be deleterious to normal tissue, the whole process should be tightly controlled. Macrophages, one type of the professional phagocytes, play a critical role in inflammation. Macrophages generate inflammatory response to lipopolysaccharide (LPS) through the TLR4/MD-2 complex. The cytoplasmic tail of TLR4 uses two sets of adaptor proteins: MAL-MyD88 at the plasma membrane and TRAM-TRIF at the endosomal membrane [2]. The MAL-MyD88 pathway triggers the initial activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK)s, resulting in the production of inflammatory proteins such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 [3,4]. NF-κB activation occurs when IκBα, which keeps NF-κB in the cytosol under resting conditions, is degraded, allowing free NF-κB to migrate to the nucleus and bind to κB sites in various inflammatory gene promoters [5]. MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)1/2) regulate the activity of activator protein 1 (AP-1) and other transcription factors necessary for inflammation or directly regulate the mRNA stability of inflammatory genes [6,7]. The TRAM-TRIF pathway leads to the activation of interferon regulatory factor (IRF)-3 and the transcription of interferon (IFN)-β and the late-phase activation of NF-κB and MAPKs [2]. Signal transducer and activator of transcription 1 (STAT1) is a latent cytosolic transcription factor that mediates INF-γ-dependent gene expression including iNOS [8–10]. IFN-γ, originally called ‘macrophage activating factor’, primes macrophages for enhanced response to TLR and amplifies TLR-induced NF-κB activation [11]. When IFN-γ binds to its receptor, the receptor-associated janus kinase (JAK)1 and JAK2 phosphorylate STAT1, leading to the dimerization of the protein, its migration to the nucleus, and binding to the DNA in target gene promoters [12]. Kudzu (Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep) is a perennial and leguminous vine native to Southeast Asia and was introduced to North America in the late 1800s to feed livestock and prevent soil erosion. Kudzu is resistant to insect pests and drought and is one of the energy crops in the US [13]. On the other hand, the growth of kudzu is so aggressive that it destroys native vegetation, giving it the status of a pest species [14]. Kudzu root has a long history of medicinal use for fever, diarrhea, diabetes, and hangover in China, Japan and Korea [15]. Not only the root but also the flower has been used for alcohol intoxication [15]. Most biological studies have been focused on the kudzu root and flower. In addition, kudzu leaves are edible and used in various foods, as are the root and flower. Kudzu leaves contain kakkalide, genistin, rutin, robinin (kaempferol-3-O-robinoside-7-O-rhamnoside), nicotiflorin (kaepmferol-3-O-rutinoside), and kaikosaponin III [16]. In this study, we investigated whether the kudzu leaf extract showed any inflammatory effects on the production of iNOS, COX-2, TNF-α, and IL-6 in macrophages and compared its efficacy with that of the kudzu root extract. Further, we tried to establish the underlying mechanism of kudzu leaf extract during stimulation with LPS or LPS plus IFN-γ. We also characterized the activity of robinin, a major constituent of kudzu leaf extract.

2. Results

2.1. Effects of Kudzu Leaf Extract on Cell Viability and the Production of Inducible Nitric Oxide Synthase (iNOS) and Nitric Oxide in Mouse Peritoneal Macrophages First, we measured the effects of kudzu leaf and root extracts on cell viability using the MTT method. Mouse peritoneal macrophages were treated with increasing concentrations of leaf or root extract. Concentrations of both types of extracts up to 400 µg/mL were not cytotoxic to peritoneal macrophages (Figure1A,B). We ﬁrst examined whether kudzu leaf extract affects LPS-induced iNOS production in peritoneal macrophages. Because we isolated peritoneal macrophages from BALB/c mice, a Th2-dominant strain, these cells require IFN-γ to express LPS-induced iNOS and nitric oxide Int. J. Mol. Sci. 2018, 19, x 1536 FOR PEER REVIEW 3 of 15

mice, a Th2-dominant strain, these cells require IFN-γ to express LPS-induced iNOS and nitric oxide (NO)(NO) productionproduction [[17].17]. Our preliminary tests showed that kudzu leafleaf extractextract completelycompletely inhibitedinhibited iNOS productionproduction atat 100100 µμg/mL.g/mL. Thus, we limited thethe maximummaximum concentrationconcentration toto 5050 µμg/mLg/mL and compared the potency of the leaf extract with that of thethe rootroot extract.extract. Decreases in the iNOS protein band were observedobserved inin cellscells treatedtreated withwith aa concentrationconcentration asas lowlow asas 1010µ μg/mLg/mL of leaf extract. The root extract also inhibited iNOS protein in a dose-depende dose-dependentnt manner, butbut the inhibitory activity of the leaf extract was much stronger than that of the root extractextract (Figure(Figure1 1C).C).Subsequently Subsequently NONO generationgeneration inin supernatant waswas measuredmeasured using using the the Griess Griess reaction. reaction. Nitrite Nitrite accumulation accumulation was was used used as as an an indicator indicator of NOof NO generation. generation. Likewise, Likewise, the the leaf leaf extract extract was was more more potent potent than than the the root root extractextract inin decreasingdecreasing nitrite accumulation (Figure1 1D).D).

Figure 1. Effects of kudzu leaf and root extracts on cell viability and the producti productionon of inducible nitric oxide synthase (iNOS) and nitric oxide (NO). ((A,,B):): MouseMouse peritonealperitoneal macrophagesmacrophages isolatedisolated fromfrom BALB/c mice mice were were cultured cultured with with kudzu ( A)) leaf leaf extract or ( B) root extract for 24 h.h. Cell viability was determined usingusing the the MTT MTT assay. assay. Data Data are representedare represented as percentages as percentages of control of control cells (0 µcellsg/mL (0 extract)μg/mL (extract)n = 4). *(np =< 4). 0.05, * p **< 0.05,p < 0.01, ** p ***< 0.01,p < 0.005*** p < vs. 0.005 control. vs. control. (C,D): ( MouseC,D): Mouse peritoneal peritoneal macrophages macrophages were stimulatedwere stimulated with LPS with (L) LPS and (L) IFN- andγ (I) IFN- in theγ (I) presence in the presence of kudzu of leaf kudzu or root leaf extract or root for extract 24 h. Whole for 24 cell h. proteinWhole cell was protein extracted was and extracted the level and of iNOS the level protein of iNOS was analyzed protein was by Western analyzed blotting by Western using GAPDHblotting asusing an internalGAPDH control. as an internal One of ﬁvecontrol. independent One of five experiments independent is shown. experiments The nitrite is shown. accumulation The nitrite in theaccumulation supernatant in wasthe supernatant measured by was the me Griessasured reagent by the assayGriess (n reagent= 3). ### assayp < ( 0.005n = 3). vs. ### control p < 0.005 (− vs.L); ***controlp < 0.005 (−L); vs.*** controlp < 0.005 (+L). vs. control (+L).

2.2. Effects of Kudzu Leaf Extract onon Cyclooxygeanse-2,Cyclooxygeanse-2, TumorTumor NecrosisNecrosis Factor- Factor-αα,, and and Interleukin-6 Interleukin-6 in in Mouse PeritonealMouse Peritoneal Macrophages Macrophages Next, we measured the effects of kudzukudzu leafleaf extractextract onon COX-2COX-2 productionproduction inin LPS-stimulatedLPS-stimulated macrophages. Cells Cells were were stimulated stimulated with with LPS LPS in in the the presence presence of ofleaf leaf or orroot root extract extract at 25, at 50, 25, and 50, and100 100μg/mL.µg/mL. The Theleaf leafextract extract was was much much more more potent potent than than the the root root extract extract in in inhibiting inhibiting COX-2 COX-2 production (Figure(Figure2 A).2A). A A concentration concentration as lowas low as25 as µ25g/mL μg/mL of leaf of extractleaf extract clearly clearly suppressed suppressed COX-2 COX-2 while higherwhile concentrationshigher concentrations (above 100(aboveµg/mL) 100 μ ofg/mL) root extractof root were extract required were required to decrease to it.decrease Finally, it. we Finally, examined we whetherexamined kudzu whether leaf kudzu extract leaf inﬂuences extract LPS-stimulated influences LPS-stimulated TNF-α and IL-6 TNF- production.α and IL-6At production. 6 h (Figure At2B,D) 6 h and(Figure 24 h2B,D) (Figure and2 C,E)24 h time(Figure points, 2C,E) the time leaf points, extract the decreased leaf extract the decreased levels of the TNF- levelsα and of TNF- IL-6α more and potentlyIL-6 more than potently did the than root did extract. the root Cells extract. treated Cell withs treated leaf or with root extractleaf or aloneroot extract did not alone produce did anynot detectableproduce any levels detectable of each levels cytokine. of each cytokine.

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FigureFigure 2.2. EffectsEffects ofof kudzukudzu leafleaf andand rootroot extractsextracts onon cyclooxygenasecyclooxygenase (COX)-2,(COX)-2, tumortumor necrosisnecrosis factorfactor (TNF)-(TNF)-αα,, and and interleukin interleukin (IL)-6 (IL)-6 in mouse in peritonealmouse peritoneal macrophages. macrophages. (A): Mouse ( peritonealA): Mouse macrophages peritoneal weremacrophages stimulated were with stimulated LPS (L) with in the LPS presence (L) in the of kudzupresence leaf of orkudzu root leaf extract or root for extract 24 h. Thefor 24 level h. The of COX-2level of protein COX-2 wasprotein analyzed was analyzed by Western by Western blotting usingblotting GAPDH using GAPDH as an internal as an control.internal Onecontrol. of three One independentof three independent experiments experiments is shown. is (B shown.–E): Mouse (B–E peritoneal): Mouse peritoneal macrophages macrophages were stimulated were stimulated with LPS (L)with for LPS 24 h, (L) and for levels 24 h, of and TNF- levelsα and of IL-6TNF- atα 6 and h (B ,IL-6D) and at 6 24 h h(B (C,D,E)) and were 24 measured h (C,E) were by enzyme-linked measured by immunosorbentenzyme-linked immunosorbent assay (ELISA) (n assay= 3).### (ELISA)p < 0.005 (n = vs. 3). control### p <( −0.005L);* vs.p < control 0.05, ** (p−L);< 0.01, * p < *** 0.05,p < 0.005** p < vs.0.01, control *** p < (+L). 0.005 vs. control (+L).

2.3.2.3. EffectsEffects ofof KudzuKudzu LeafLeaf ExtractExtract onon NuclearNuclear Factor-Factor-κκB,B, Mitogen-ActivatedMitogen-Activated ProteinProtein Kinase,Kinase, and and TABK- TABK-BindingBinding Kinase Kinase 1 Activation 1 Activation in Mouse in Mouse Peritoneal Peritoneal Macrophages Macrophages WeWe investigatedinvestigated any effect effect of of kudzu kudzu leaf leaf extract extract on on the the degradation degradation of I ofκB IακB, aα critical, a critical step step in NF- in NF-κB κandB and signaling signaling [18]. [18 Cells]. Cells stimulated stimulated with with LPS LPS for for 15 15 min min showed showed complete complete degradation ofof IIκκBBαα andand kudzukudzu leafleaf extractextract diddid notnot inﬂuenceinfluence thisthis processprocess (Figure(Figure3 3A).A). We We also also examined examined whether whether kudzu kudzu leafleaf extractextract affectsaffects NF-NF-κκBB transcriptionaltranscriptional activityactivity usingusing luciferaseluciferase reporterreporter assays.assays. KudzuKudzu leafleaf extractextract alonealone diddid notnot stimulatestimulate NF-NF-κκBB activityactivity (Figure(Figure S1).S1). LPSLPS increasedincreased NF-NF-κκBB transcriptionaltranscriptional activityactivity andand kudzukudzu leafleaf extractextract diddid notnot showshow anyany inhibitoryinhibitory effectseffects (Figure(Figure3 3B).B). We We examined examined MAPK MAPK activation activation andand AP-1AP-1 transcriptional activity. Stimulation Stimulation with with LPS LPS for for 15 15 min min caused caused the the phosphorylation phosphorylation of ofJNK, JNK, p38, p38, andand ERK1/2 ERK1/2 (Figure (Figure 3A). Among3A). Among these MA thesePKs, MAPKs, kudzu leaf kudzu extract leaf decreased extract decreased the expression the of phosphorylated JNK but rather enhanced the transcriptional activity of AP-1 (Figure 3C). We investigated whether kudzu leaf extract may interfere with TANK-binding kinase 1 (TBK1)

Int. J. Mol. Sci. 2018, 19, 1536 5 of 15 expressionInt. J. Mol. Sci. of 2018 phosphorylated, 19, x FOR PEER JNKREVIEW but rather enhanced the transcriptional activity of AP-1 (Figure 53 ofC). 15 We investigated whether kudzu leaf extract may interfere with TANK-binding kinase 1 (TBK1) phosphorylationphosphorylation asas aa measuremeasure ofof TRIF signaling. Kudzu leaf extract decreased TBK1 phosphorylation (Figure(Figure3 3A).A).Interestingly, Interestingly, it it also also decreased decreased TBK1 TBK1protein proteinexpression expression inin aadose-dependent dose-dependent manner,manner, indicatingindicating thatthat thethe decreasedecrease inin TBK1TBK1 phosphorylationphosphorylation by kudzu leaf extractextract waswas mediatedmediated by itsits decreasedecrease inin TBK1TBK1 proteinprotein itself.itself.

Figure 3. Effects of kudzu leaf extract on nuclear factor-κB (NF-κB), mitogen-activated protein kinase Figure 3. Effects of kudzu leaf extract on nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), activator protein 1 (AP-1), and Tank-binding kinase 1 (TBK1) activation. (A) Mouse peritoneal (MAPK), activator protein 1 (AP-1), and Tank-binding kinase 1 (TBK1) activation. (A) Mouse macrophages were treated with kudzu leaf extract for 1 h followed by stimulation with LPS (L) for peritoneal macrophages were treated with kudzu leaf extract for 1 h followed by stimulation with 15 min. Whole cell extracts were prepared and subjected to Western blotting. The level of signaling LPS (L) for 15 min. Whole cell extracts were prepared and subjected to Western blotting. The level of proteins was analyzed using GAPDH as an internal control. One of three independent experiments signaling proteins was analyzed using GAPDH as an internal control. One of three independent is shown. (B,C) RAW264.7 cells were transfected with an NF-κB-or AP-1-dependent reporter gene. experiments is shown. (B,C) RAW264.7 cells were transfected with an NF-κB-or AP-1-dependent Cells were pretreated with kudzu leaf extract for 2 h and then stimulated with LPS for 6 h. Luciferase reporter gene. Cells were pretreated with kudzu leaf extract for 2 h and then stimulated with LPS for activity was measured using the Dual-Glo® luciferase assay system (n = 3). ### p < 0.005 vs. control 6 h. Luciferase activity was measured using the Dual-Glo® luciferase assay system (n = 3). ### p < 0.005 (−L); *** p < 0.005 vs. control (+L). vs. control (−L); *** p < 0.005 vs. control (+L).

2.4.2.4. Effects of Kudzu Leaf Extract on Signal TransducerTransducer andand ActivatorActivator ofof TranscriptionTranscription 1 1 Activation Activation in in Mouse Peritoneal Macrophages Mouse Peritoneal Macrophages Because the strong inhibition of iNOS production by kudzu leaf extract did not appear to be Because the strong inhibition of iNOS production by kudzu leaf extract did not appear to be related to NF-κB and AP-1, we investigated whether this reduction may be associated with the STAT1 related to NF-κB and AP-1, we investigated whether this reduction may be associated with the STAT1 pathway that is required for iNOS expression [9,10]. Cells were stimulated with LPS and IFN-γ to pathway that is required for iNOS expression [9,10]. Cells were stimulated with LPS and IFN-γ to maximize STAT1 activation [8]. We measured phosphorylation of STAT1 on tyrosine 701. Kudzu leaf maximize STAT1 activation [8]. We measured phosphorylation of STAT1 on tyrosine 701. Kudzu leaf extract strongly inhibited STAT1 phosphorylation in a dose-dependent manner: STAT1 activation was extract strongly inhibited STAT1 phosphorylation in a dose-dependent manner: STAT1 activation abrogated at 100 µg/mL kudzu leaf extract (Figure4). Notably, decreases in STAT1 were observed at was abrogated at 100 μg/mL kudzu leaf extract (Figure 4). Notably, decreases in STAT1 were increasing concentrations of kudzu leaf extract. observed at increasing concentrations of kudzu leaf extract.

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FigureFigure 4. 4. EffectEffect of of kudzu kudzu leaf leaf extract extract on on signal signal tran transducersducer and and activator activator of of transcription transcription 1 1(STAT1) (STAT1) activation.activation. Mouse Mouse peritoneal peritoneal macrophages macrophages were were trea treatedted with with kudzu kudzu leaf leaf extract extract for for 1 1h hfollowed followed by by stimulationstimulation with with LPS LPS (L) (L) and and IFN- IFN-γγ (I)(I) for for 22 h. h. Whole Whole cell cell extracts extracts were were prepared prepared and and subjected subjected to to WesternWestern blotting. blotting. The The level level of of signaling signaling proteins proteins wa wass analyzed analyzed using using GAPDH GAPDH as as an an internal internal control. control. OneOne of of three three independent independent experiments experiments is is shown. shown.

2.5.2.5. High-performance High-performance Liquid Liquid Chromatography Chromatography (HPLC) (HPLC) Analysis Analysis of of Robinin Robinin and and Puerarin Puerarin in in Kudzu Kudzu Leaves Leaves andand Roots Roots AA comparison comparison of of the the contents contents of of genistein, genistein, genistin, genistin, daidzein, daidzein, daidzin, daidzin, and and puerarin puerarin between between kudzukudzu leaf leaf and and root root was was reported reported [19]: [19]: the the leaf leaf contained contained 1/10 1/10 to to 1/40 1/40 of of these these isoflavonoids isoflavonoids compared compared withwith the the root. root. Here, Here, we we chose chose robinin robinin and and puerarin, puerarin, the the known known major major isoflavone isoflavone in in kudzu kudzu root, root, and and comparedcompared their their contents. contents. In In the the high-performance high-performance liquid liquid chromatography chromatography (HPLC) (HPLC) analysis analysis of of leaf leaf extracts,extracts, one one major major substance substance was was detected detected at at a arete retentionntion time time of of 10.2 10.2 min min on on the the 350 350 nm nm ultraviolet ultraviolet (UV)-spectrum(UV)-spectrum (Figure (Figure 5A).5A). The The compound compound was was confirmed confirmed as as robinin robinin by by photodiode photodiode array array spectral spectral 1 13 analysisanalysis of of HPLC HPLC using using λλmaxmax = =265.0 265.0 and and 347.1 347.1 nm nm values values and and also also by by 1H-H- and and 13C-NMRsC-NMRs (Figure (Figure S2) S2) afterafter the the isolation isolation process process of of the the compound. compound. Robinin Robinin showed showed a apurity purity of of 90%, 90%, based based on on the the peak peak area area atat 350 350 nm nm detection detection of of HPLC HPLC chromatogram chromatogram (Figure (Figure 55CC).). PuerarinPuerarin inin thetheroot root extract extract was was detected detected at at a aretention retention time time of of 8.2 8.2 min min on on the the 250 250 nm nm UV-spectrum, UV-spectrum, exhibiting exhibitingλmax λmax= 249.7= 249.7 and and 304.2 304.2 nm nm (Figure (Figure5B). 5B).The The compound compound was was confirmed confirmed by HPLC by HPLC analysis analysis of standard of standard puerarin puerarin (Figure (Figure5D).The 5D). yields The yields of the ofmethanol the methanol extracts extracts from thefrom leaves the leaves and roots and wereroots about were 11%about and 11% 25%, and respectively 25%, respectively (Table1 ).(Table Robinin 1). Robininwas present was present in about in 0.46% abou oft 0.46% the dry of weightthe dry ofweight leaf extract of leaf and extr wasact and not was detected not detected in the roots in the (Figure roots3 , (FigureTable1 ).3, InTable contrast, 1). In puerarincontrast, waspuerarin abundantly was abundantly present in present about 4.35%in about of 4.35% the root of drythe root weight dry and weight was andnot was detected not detected in the leaf in extract.the leaf Thisextract. pattern This waspattern similar was tosimilar the content to the ofcontentPueraria of Pueraria montana montanareported reportedby Kirakosyan by Kirakosyan et al. [19 et], inal. which [19], in the which puerarin the pu levelerarin in thelevel roots in the showed roots showed the highest the value,highest while value, far whilelower far amounts lower amounts of puerarin of puerarin were found were in found the leaves in the and leaves other and organs. other organs.

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FigureFigure 5. 5. HPLCHPLC chromatograms ofof kudzukudzu leaf leaf extract extract (A (A),), root root extract extract (B ),(B robinin), robinin (C ),(C and), and puerarin puerarin (D). (D). Table 1. Contents of major ﬂavonoids in kudzu leaf and root. All units: mg/g dry weight. Table 1. Contents of major flavonoids in kudzu leaf and root. All units: mg/g dry weight. Robinin Puerarin Yield of Crude Extract Robinin Puerarin Yield of Crude Extract Leaf 4.61 ± 0.18 N.D. 110.2 RootLeaf 4.61N.D. ± 0.18 43.54 N.D.± 0.83 110.2 253.4 Root N.D. 43.54N.D. “not ± 0.83 detected”. 253.4 N.D. “not detected”. 2.6. Effects of Robinin on iNOS Production and Signaling Molecules in Activated Macrophages 2.6. Effects of Robinin on iNOS Production and Signaling Molecules in Activated Macrophages Because robinin exists abundantly in kudzu leaf, we examined whether this compound might affectBecause the iNOS robinin protein exists levels abundantly in activated in macrophages.kudzu leaf, we We examined conﬁrmed whether that robinin this compound concentrations might up affectto 100 theµM iNOS did notprotein show levels any cytotoxicity in activated (Figure macrophages.6A). Robinin We confirmed decreased that LPS/IFN- robininγ -inducedconcentrations nitrite upand to iNOS 100 productionμM did not in show a dose-dependent any cytotoxicity manner (Figure (Figure 6A).6B,C). Robinin However, decreased LPS-induced LPS/IFN- productionγ-induced of nitriteTNF-α andat 6 iNOS and 24 production h was unaltered in a dose-depende (Figure6D,E). Nont manner change in(Figure COX-2 6B,C). and IL-6 However, was observed LPS-induced (Figure productionS3). Robinin of itselfTNF- didα at not 6 and stimulate 24 h was NF- unalteredκB transcriptional (Figure 6D,E). activity No (Figurechange S1)in COX-2 but rather and enhancedIL-6 was observedLPS-induced (Figure NF- κS3).B transcriptional Robinin itself activitydid not without stimulate effects NF- onκB ItranscriptionalκBα degradation activity (Figure (Figure7A,B). RobininS1) but ratherinhibited enhanced JNK activation LPS-induced but enhancedNF-κB transcriptional the transcriptional activity activity without AP-1 effects (Figure on I7κA,C).Bα degradation A robinin (Figureconcentration 7A,B). ofRobinin 50 µM inhibitedinhibited STAT1JNK activation phosphorylation but enhanced (Figure8 the). transcriptional activity AP-1 (Figure 7A,C). A robinin concentration of 50 μM inhibited STAT1 phosphorylation (Figure 8).

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FigureFigure 6. Effect 6. Effect of robininof robinin on on inﬂammatory inflammatory responsesresponses in mouse mouse peritoneal peritoneal macrophages. macrophages. (A) (MouseA) Mouse Figure 6. Effect of robinin on inflammatory responses in mouse peritoneal macrophages. (A) Mouse peritonealperitoneal macrophages macrophages were were cultured cultured with with robininrobinin for 24 h. h. Cell Cell viability viability was was determined determined using using the the peritoneal macrophages were cultured with robinin for 24 h. Cell viability was determined using the MTTMTT assay. assay. Data Data are are represented represented as as percentage percentage ofof controlcontrol cells (0 (0 μµg/mLg/mL robinin) robinin) (n (=n 4).= 4).(B,C (B) Cells,C) Cells MTTwere assay.stimulated Data withare represented LPS (L) and as IFN- percentageγ (I) in ofthe control presence cells of (0 robinin μg/mL forrobinin) 24 h. (Then = 4).level (B ,ofC) iNOSCells were stimulated with LPS (L) and IFN-γ (I) in the presence of robinin for 24 h. The level of iNOS protein wereprotein stimulated (B) was withanalyzed LPS by(L) Westernand IFN- blottingγ (I) in theusing presence tubulin of as robinin an internal for 24 control.h. The levelOne ofof iNOSthree (B) was analyzed by Western blotting using tubulin as an internal control. One of three independent proteinindependent (B) was experiments analyzed isby shown. Western The blotting levels of using nitrite tubu (C) werelin as measured an internal using control. the Griess One reaction.of three experiments is shown. The levels of nitrite (C) were measured using the Griess reaction. (D,E) The independent(D,E) The levels experiments of TNF-α is at shown. 6 h (D) The and levels 24 h (ofE) nitritein the (supernatantC) were measured was measured using the by Griess ELISA reaction. (n = 3). α levels(###D of,E p) TNF- <0.005The levels vs.at 6control of h TNF- (D) ( and−αL); at 24***6 h hp ( (D

Figure 7. Effects of robinin on LPS-induced NF-κB and MAPK activation in mouse peritoneal FigureFiguremacrophages. 7. 7.Effects Effects (A of ) ofMouse robinin robinin peritonealon on LPS-inducedLPS-induced macrophages NF- κκwereBB and and trea MAPK MAPKted with activation activation robinin infor inmouse 1 mouseh followed peritoneal peritoneal by macrophages.macrophages.stimulation with (A ()A Mouse)LPS Mouse (L) peritoneal forperitoneal 15 min. macrophagesmacrophagesWhole cell extracts were were treawere treatedted pr withepared with robinin robininand subjectedfor for1 h 1followed hto followedWestern by by stimulation with LPS (L) for 15 min. Whole cell extracts were prepared and subjected to Western stimulationblotting. withThe level LPS of (L) signaling for 15 min.proteins Whole was analyz cell extractsed using were GAPDH prepared as an andinternal subjected control. toOne Western of blotting. The level of signaling proteins was analyzed using GAPDH as an internal control. One of blotting.three independent The level of experiments signaling proteins is shown. was (B, analyzedC): RAW264.7 using cells GAPDH were transfected as an internal with an control. NF-κB One(B) of three independent experiments is shown. (B,C): RAW264.7 cells were transfected with an NF-κB (B) threeor independent AP-1 (C)-dependent experiments reporter is gene. shown. Cells (B were,C): RAW264.7pretreated with cells robinin were transfected for 2 h and then with stimulated an NF-κB( B) orwith AP-1 LPS (C for)-dependent 6 h. Luciferase reporter activi gene.ty was Cells measured were pretreat usinged the with Dual-Glo robinin® luciferasefor 2 h and assay then systemstimulated (n = or AP-1 (C)-dependent reporter gene. Cells were pretreated with robinin® for 2 h and then stimulated with3). ### LPS p <0.005 for 6 h.vs. Luciferase control (− L);activi ***ty p

FigureFigure 8. 8. EffectEffect of of robinin robinin on on LPS/IFN- LPS/IFN-γ-inducedγ-induced STAT1 STAT1 activation activation in in mouse mouse peritoneal peritoneal macrophages. macrophages. MouseMouse peritoneal peritoneal macrophages macrophages were were treated with robininrobinin forfor 11h h followed followed by by stimulation stimulation with with LPS LPS (L) (L)and and IFN- IFN-γ (I)γ (I) for for 2 h. 2 Wholeh. Whole cell cell extracts extracts were were prepared prepared and and subjected subjected to Western to Western blotting. blotting. The The level levelof signaling of signaling proteins proteins was analyzed was analyzed usingGAPDH using GAPDH as an internal as an control. internal One control. of three One independent of three independentexperiments experiments is shown. is shown.

3.3. Discussion Discussion InIn comparison comparison to to kudzu kudzu root root and and flower, flower, which which have have a along long history history of of medical medical use, use, little little attention attention hashas been been paid paid to to kudzu kudzu leaf. leaf. In In this this sense, sense, our our study study shed shed light light on on the the importance importance of of kudzu kudzu leaf leaf as as a a richrich source source of of anti-inflammatory anti-inflammatory constituents. constituents. Kudzu Kudzu leaf leaf extract extract was was much much more more potent potent than than its its root root extractextract in in inhibiting inhibiting the the important important inflammatory inflammatory markers markers such such as as iNOS, iNOS, COX-2, COX-2, TNF- TNF-αα, and, and IL-6 IL-6 in in macrophagesmacrophages stimulated stimulated wi withth LPS LPS or or LPS LPS plus plus IFN- IFN-γγ. . InIn macrophages, macrophages, iNOS iNOS catalyzes catalyzes the the conversion ofof L-arginine,L-arginine, molecularmolecular oxygen, oxygen, and and NADPH NADPH to toL -citrullin,L-citrullin, water, water, and and NO NO [20]. [20]. NO reactsNO reacts with the with thiol the groups thiol ofgroups cysteins of andcysteins forms andS-nitrosothiols, forms S- nitrosothiols,altering protein altering function protein and reactingfunction with and superoxidereacting with to become superoxide the more to become toxic peroxynitrite, the more toxic thus peroxynitrite,causing oxidation thus causing and DNA oxidation damage and [20]. DNA COX-2 damage catalyzes [20]. the COX-2 conversion catalyzes of arachidonicthe conversion acid of to arachidonicprostaglandin acid H to2, whichprostaglandin leads to H the2, formationwhich leads of to various the formation prostanoids of various [21]. Prostagladin prostanoids E 2[21]., one Prostagladintype of prostanoids, E2, one type elicits of vasodilation,prostanoids, vascularelicits vasodilation, hyperpermeability, vascularfever, hyperpermeability, and pain. TNF- fever,α and and IL-6 pain.activate TNF- endothelialα and IL-6 cells activate and otherendothelial leukocytes cells andand induceother leukocytes the acute-phase and induce response the [ 1acute-phase]. Therefore, responsethese inflammatory [1]. Therefore, proteins these areinflammatory molecular targets proteins for are the molecular development targets of anti-inflammatory for the development agents. of anti-inflammatoryMany inflammatory agents. genes such as TNF-α, IL-6, COX-2, and iNOS are co-dependent on MyD88Many and inflammatory TRIF but the genes precise such role as TNF- of theα,two IL-6, pathways COX-2, and in expressingiNOS are co-dependent these genes seemson MyD88 to be anddistinct TRIF [but4,22 the]. For precise example, role of LPS-induced the two pathways TNF-α inproduction expressing is these impaired genes in seem MyD88-ors to be distinct TRIF-deficient [4,22] Formacrophages example, LPS-induced [22,23]. However, TNF-α the production MyD88 pathway is impaired upregulates in MyD88-or TNF-α TRIF-deficientgene via activation macrophages of NF-κB [22,23].while theHowever, TRIF pathway the MyD8 regulates8 pathway TNF- upregulatesα promoter TNF- activityα gene with via little activation dependence of NF- onκB NF-whileκB[ the24 ]. TRIFSeveral pathway studies regulates indicate thatTNF- theα contributionpromoter activity of the TRAM-TRIFwith little dependence pathway to on NF- NF-κB signalingκB [24]. Several is minor studiesand delayed indicate relative that the to thecontribution MyD88 pathway of the TRAM-TRIF [3,24,25]. Our pathway data showed to NF- thatκB signaling the MyD88-dependnet is minor and delayedNF-κB andrelative AP-1 to transcriptional the MyD88 pathway activity [3,24,25]. was the leastOur data likely showed target ofthat kudzu the MyD88-dependnet leaf extract. Instead, NF- the κsuppressiveB and AP-1 effecttranscriptional of kudzu leafactivity extract was on the TBK1 least phosphorylation, likely target of upstreamkudzu leaf of extract. IRF-3 activation, Instead, maythe suppressiveaccount for effect one possible of kudzu mechanism leaf extract involving on TBK1 the phosphorylation, TRIF pathway that upstream mediates of inflammatoryIRF-3 activation, response may accountindependently for one ofpossible NF-κB andmechanism AP-1. Many involving inflammatory the TRIF genes pathway including that mediates TNF-α, IL-6, inflammatory and COX-2 responsecontain AU-richindependently elements of (ARE)NF-κB inand the AP-1. 30-untranslated Many inflamma regions,tory which genes determines including TNF- the rateα, IL-6, of mRNA and COX-2stability contain and translation AU-rich elements [26]. This (ARE) sequence in the is regulated3′-untranslated by various regions, ARE-binding which determines proteins. the In additionrate of mRNAto the modulationstability and of translation AP-1 and NF-[26].κ B,This MAPKs sequence participate is regulated in the by inflammatory various ARE-binding response byproteins. regulating In additionthe stability to the of modulation cytokine genes of AP-1 at the and post-transcriptional NF-κB, MAPKs participate level. JNK isin required the inflammatory for LPS-induced response TNF- byα regulatingand iNOS the mRNA stability translation of cytokine [27,28 ].genes It is possibleat the post-transcriptional that without the involvement level. JNK of is AP-1 required transcriptional for LPS- inducedactivity, TNF- kudzuα and leaf iNOS extract mRNA may affecttranslation theeinflammatory [27,28]. It is possible gene mRNA that without stability the via involvement modulation of of AP-1JNK transcriptional activation. activity, kudzu leaf extract may affect thee inflammatory gene mRNA stability via modulationAmong the of inflammatoryJNK activation. proteins tested here, iNOS responded more sensitively to kudzu leaf extractAmong than the the inflammatory other inflammatory proteins markers. tested Moreover,here, iNOS the responded suppressive more effect sensitively of kudzu to leaf kudzu extract leaf on extract than the other inflammatory markers. Moreover, the suppressive effect of kudzu leaf extract on STAT1 activation was remarkable. Because STAT1 is required for iNOS expression in

Int. J. Mol. Sci. 2018, 19, 1536 10 of 15

STAT1 activation was remarkable. Because STAT1 is required for iNOS expression in macrophages, the inhibition of iNOS and STAT1 by kudzu leaf extract might be correlated. In addition to its direct involvement of iNOS expression, STAT1 contributes to other TLR-induced inflammatory responses: peritoneal macrophages from STAT1 deficient mice showed lower levels of LPS-induced TNF-α and IL-6 production compared to those from wild type mice [29]. This is due to the fact that crosstalk between IFNs and TLR signaling pathways occurs at multiple levels [12,30]. In response to LPS, macrophages activate themselves in an autocrine and paracrine manner by producing IFN-α/β and, to a lesser degree, IFN-γ [31]. IFN-α/β and IFN-γ share the JAK/STAT1 pathway [8]. Thus, inhibition of STAT1 by kudzu leaf extract may confer its anti-inflammatory effects by giving negative feedback to LPS-induced signaling. An interesting observation is that kudzu leaf extract decreases the level of STAT1 protein in a dose-dependent manner. Therefore, the observed inhibitory effect of kudzu leaf extract on STAT1 activation was partially due to an altered level of STAT1 protein. We do not know the mechanism by which kudzu leaf extract causes the degradation of STAT1. Macrophages from IFN-β(−/−) mice showed less STAT1 protein than macrophages from wild type mice [32]. Whether the decrease in STAT1 by kudzu leaf extract is associated with IFN-β signaling remains to be determined. An analysis of the quantities of isoflavonoids in each organ of kudzu showed that the root contains much higher levels of medicinally important isoflavonoids such as daidzein, daidzin, genistein, genistin, and puerarin than do its aerial parts including leaves [19]. Daidzein, genistein, and puerarin are reported to decrease LPS-induced iNOS production in macrophages [33,34]. A comparative study on the effects of various flavonoids demonstrated that genistein decreased TNF-α production in LPS/IFN-γ-activated macrophages while daidzein, daidzin, and genistin increased its production in a dose-dependent manner [35]. Although kudzu leaves are not reported to be a good source of the known beneficial flavonoids, they seem to contain unidentified bioactive components that exert anti-inflammatory activity. Given the amount of robinin contained in the effective dose of kudzu leaf extract (100 µg/mL of kudzu leaf extract contains about 0.46 µg/mL or 0.6 µM) and its lack of some biological activities such as TNF-α reduction, the anti-inflammatory effect of kudzu leaf is clearly not dependent on robinin. Robinin is found in other plants such as some species from the Robinia and Astragalus genera and Vinca erecta [36,37]. Robinin is one of the major active components of Flaronin, which is a pharmaceutical product prepared from the leaves and flowers of Astragalus falcatus L. in Russia and Georgia and has been shown to enhance the nitrogen-excretory function of kidneys and increase diuresis [37]. Kaempferol, the parent aglycone of robinin, is one of the most common dietary flavonoids and inhibits the expression of iNOS, COX-2, and TNF-α through its modulation of NF-κB, AP-1, and STAT1 in LPS-stimulated macrophages [33,38]. Generally, the potency of glycosides has been demonstrated to be weaker than that of their aglycones because they are less penetrable to cell membranes. We found that kaempferol was much more potent in inhibiting iNOS production than was robinin (data not shown). The downregulation of iNOS by robinin seemed to be mediated by its inhibition of JNK and STAT1. Robinin can be hydrolyzed to kaempferol by intestinal microflora [39]. This suggests that robinin might exert a more potent inhibitory effect when it is consumed orally and converted to kaempferol in the gut. Taken together, we discovered that despite being low in content of the known anti-inflammatory isoflavonoids, kudzu leaves showed more potent anti-inflammatory activity in macrophages than did kudzu roots. Kudzu leaf extract suppressed LPS-induced production of iNOS, COX-2, TNF-α, and IL-6 via modulation of JNK, TBK1, and STAT1 activation. We also identified that robinin, a major component of kudzu leaf, suppressed iNOS production through the modulation of JNK and STAT1 activity. However, robinin did not represent the anti-inflammatory activity of kudzu leaf extract. Our data provide evidence that in addition to the use of kudzu root as a medicinal and food resource, kudzu leaf is an excellent source of as yet unknown anti-inflammatory constituents. Int. J. Mol. Sci. 2018, 19, 1536 11 of 15

4. Materials and Methods

4.1. Sample Preparation Leaves and roots of kudzu were harvested at the boundary hill between an agricultural field and mountain in Yongin, Korea in July 2014. The fully expanded young leaves were chosen, and the tap roots were selected by removal of the fine roots. After being rinsed three times with distilled water, the leaves and sliced roots (3 mm in thickness) were dried in a 38 ◦C dry oven for five days and ground with a commercial home grinder. The samples were stored at −20 ◦C until extraction.

4.2. Extraction Procedure Leaf or root power (each 750 g) was divided equally into three groups. Each sample (250 g) was suspended in 5 L of methanol and kept in a 4 ◦C cold chamber for 48 h for extraction. Experiments were carried out in triplicate. The suspension was collected after a ﬁltering process, and the debris was suspended again in 5 L of methanol. These steps were repeated until the solvent color disappeared completely to the naked eye. After pooling the suspensions, a vacuum reﬂux cooling system in a rotary evaporator (N-1100, Eyelar Co., Tokyo, Japan) was applied to evaporate the solvent from the extracts. The samples were then freeze-dried. For cell culture, these extracts were dissolved in dimethyl sulfoxide (DMSO).

4.3. Isolation of Robinin The solvent fractionations using the principle of solvent polarity were performed with n-hexane and ethylacetate. Leaf extracts (30 g) attained by the procedure of methanol extraction were dissolved into 1 L of distilled water set into a fraction funnel (3 L volume size) and 1 L of n-hexane. The complex solvents were shaken strongly and kept silently for 30 min. After stabilization of the fraction reaction, the n-hexane fraction was removed from the water-dissolved extract. After repeating the procedure of n-hexane fractionation, further fractionation using ethylacetate with the same procedure as that for n-hexane fractionation was performed. A compound that was crystallized during the ethylacetate fractionation in water-dissolved leaf extract was separated by ﬁltering and several washing processes.

4.4. HPLC Analysis Puerarin, the isolated compound, and each extract of leaf and root were dissolved in methanol to a concentration of 0.5 mg/mL, and filtered with a syringe filter (0.45 µm, Futecs, Daejeon, Korea). The HPLC procedure used here was performed similarly to that described by Seo et al. [40], except for the running combination of the mobile phases, solvent A (0.1% formic acidic water) and solvent B (0.1% formic acidic acetonitrile). The mobile phases ran at 15% solvent B for 0–5 min, 20% solvent B for 5–15, 30% solvent B for 15–20 min, and 35% solvent B for 20–23 min. The HPLC system was a Waters 2695 separation module (Waters, Milford, MA, USA) with a Waters 996 photodiode array detector set to 350 nm for robinin and 250 nm for puerarin. The column was a 250 × 4.6 mm Prontosil HPLC column (120-5-C18-ace-EPS 0.5 µm, Bischoff, Snackenberg, Germany). The sample injection volume was 10 µL. The flow rate was 0.8 mL/min, and the column temperature was kept at 30 ◦C.

4.5. Isolation of Mouse Peritoneal Macrophages Seven-week-old male BALB/c mice were obtained from DBL (Eumsung, Korea) and housed in a temperature- and humidity-controlled pathogen-free animal facility with a 12-h light-dark cycle. The animal protocol (KHUASP(SE)-15-012) was approved by our institutional committee, and mice were cared for according to the US National Research Council for the Care and Use of Laboratory Animals speciﬁcations (1996). Mice were injected intraperitoneally with 2 mL of 3.5% sterile thioglycollate solution (BD, Sparks, MD, USA), and four days later, mice were sacriﬁced by cervical dislocation. Peritoneal exudate cells were isolated by peritoneal lavage with cold DMEM Int. J. Mol. Sci. 2018, 19, 1536 12 of 15

(Hyclone, Logan, UT, USA). After centrifugation, cells were resuspended in DMEM with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin. Cells were plated overnight at 37 ◦C, and non-adherent cells were removed.

4.6. Cell Viability Assay Cells were seeded in quadruplicate in 96-well plates overnight and the culture medium was removed. Wells were replenished with new medium containing experimental samples for 24 h, and then cell viability was determined using the MTT assay. Brieﬂy, the culture medium was removed and the cells were washed with phosphate buffered saline (PBS). Then, MTT (Sigma, St. Louis, MO, USA) dissolved in DMEM was added to each well to attain a ﬁnal concentration of 0.5 mg/mL. After 1 h of incubation at 37 ◦C, the media were removed and 0.1 mL DMSO was added and incubated for 15 min to solubilize the MTT. Optical density was measured at 570 nm with an iMark microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was expressed as a percentage of control cells.

4.7. Stimulation of Cells with LPS or LPS Plus IFN-γ For iNOS, NO, COX-2, and cytokine analysis, mouse peritoneal macrophages were stimulated with 100 ng/mL LPS (Sigma, St. Louis, MO, USA) plus 0.5 ng/mL IFN-γ (BD Pharmingen, San Diego, CA, USA) or LPS alone for 24 h, and samples were added simultaneously. For signaling molecule analysis, mouse peritoneal macrophages were pretreated with samples for 1 h and then stimulated with LPS for 15 min or LPS plus IFN-γ for 2 h.

4.8. Determination of Nitrite Accumulation Supernatant from the above culture was incubated with an equal volume of Griess reagent (Sigma, St. Louis, MO, USA) for 15 min at room temperature. Optical density was measured at 540 nm with the microplate reader.

4.9. Cytokine Analysis Levels of TNF-α and IL-6 were determined by enzyme-linked immunosorbent assay according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA).

4.10. Western Blot Analysis Cells were rinsed in cold PBS and then lysed on ice in 0.1 mL of RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 20 mM NaF; 0.5% NP-40; and 1% Triton X-100) containing a phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) and a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). After centrifugation at 13,000× g for 10 min, supernatants were collected. Protein concentrations were determined using the Bradford protein assay reagent (Bio-Rad, Hercules, CA, USA), and the samples were diluted with sodium dodecyl sulfate (SDS) buffer and boiled for 3 min. The samples were separated on an 8% or 10% SDS-polyacrylamide gel and were transferred to polyvinylidene ﬂuoride membranes. The membranes were then blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. The membranes were incubated with iNOS, IκBα, GAPDH, or tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX-2 (Cayman, Ann Arbor, MI, USA), phospho-JNK (T183/Y185), JNK, phospho-ERK1/2 (T202/T204), ERK, phospho-p38 (T180/Y204), p38, phospho-TBK1(S172), TBK1, phospho-STAT1 (Y701), and STAT1 (Cell Signaling Technology, Beverly, MA, USA) diluted in 5% skim milk in TBST overnight at 4 ◦C. The blots were washed with TBST and incubated for 1 h with anti-rabbit horseradish peroxidase-conjugated antibodies. Immunoreactive bands were detected with EzWestLumi plus (ATTO, Tokyo, Japan) and analyzed using an EZ-Capture MG (ATTO, Tokyo, Japan). Int. J. Mol. Sci. 2018, 19, 1536 13 of 15

4.11. Luciferase Assay NF-κB and AP-1 transcriptional activity was measured using RAW264.7 cells transfected with the pGL4.32 containing five copies of an NF-κB response element or pGL4.44 containing six copies of an AP-1 responsive element and the firefly luciferase reporter gene (luc2P) (Promega, Madison, WI, USA). The transfected RAW264.7 cells were plated to 96 well plates overnight. After a media change, cells were pretreated with robinin for 2 h and then stimulated with LPS for 6 h. Luciferase activity was measured using the Dual-Glo® luciferase assay system (Promega, Madison, WI, USA).

4.12. Statistical Analysis Data were analyzed by student t test or ANOVA followed by the Tukey test using IBM SPSS 22 software. p Values less than 0.05 were considered signiﬁcant.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/5/ 1536/s1. Author Contributions: S.-H.E. and H.K. wrote the manuscript. S.-J.J., H.-Y.J., and Y.-J.S. performed cell culture and protein analysis. Y.-J.L. performed HPLC. Y.-H.L. and J.-I.K. provided reagents. Acknowledgments: This research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the High Value-added Food Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (116005-3). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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