p‑Coumaric Acid and Ursolic Acid from Corni fructus Attenuatedβ‑Amyloid25−35-Induced Toxicity through Regulation of the NF-κBSignaling Pathway in PC12 CellsJeong-Hyun Yoon,†,∥ Kumju Youn,†,∥ Chi-Tang Ho,‡ Mukund V. Karwe,‡ Woo-Sik Jeong,§

and Mira Jun*,†

†Department of Food Science and Nutrition, Dong-A University, Busan 604-714, Korea‡Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States§Department of Food and Life Science, College of Biomedical Science and Engineering, Inje University, Gimhae 621-749, Korea

ABSTRACT: Neuroinflammatory responses induced by amyloid-beta peptide (Aβ) are important causes in the pathogenesis ofAlzheimer’s disease (AD). Blockade of Aβ has emerged as a possible therapeutic approach to control the onset of AD. This studyinvestigated the neuroprotective effects and molecular mechanisms of p-coumaric acid (p-CA) and ursolic acid (UA) from Cornifructus against Aβ25−35-induced toxicity in PC12 cells. p-CA and UA significantly inhibited the expression of iNOS and COX-2 inAβ25−35-injured PC12 cells. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NF-κB) andphosphorylation of IκB-α was also observed after p-CA and UA treatment. For the upstream kinases, UA exclusively reducedERK1/2, p-38, and JNK phosphorylation, but p-CA suppressed ERK1/2 and JNK phosphorylation. Both compoundscomprehensively inhibited NF-κB activity, but possibly with different upstream pathways. The results provide new insight intothe pharmacological modes of p-CA and UA and their potential therapeutic application to AD.

KEYWORDS: Alzheimer’s disease, amyloid β peptide, Corni fructus, p-coumaric acid, ursolic acid, COX-2, iNOS, NFκB, IκB, MAPKs

■ INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disordercharacterized by progressive loss of neurons in the brain, whichcorrelates with deposition of neurofibrillary tangles andaccumulation of senile plaques, the two pathological hallmarksof the disease.1 β-Amyloid (Aβ) peptide, a 40−42 residue of theinternal peptide segment from amyloid precursor protein(APP), is the major component of senile plaques and has beenconsidered to play a pivotal role in the development andprogress of AD.2

Aβ is liberated by the sequential actions of two proteases, β-secretase (BACE1) and γ-secretase. There is compellingevidence indicating that extracellular deposits of Aβ mayinduce activation of astrocytes and microglial cells by releasingpro-inflammatory factors that contribute to the neurodegenera-tion process in AD.3 Various epidemiological studies haveproved that the use of conventional NSAIDs can prevent and/or retard AD, which supported the notion that the neuro-inflammatory responses can be an attractive target fortreatment with anti-inflammatory drugs.4 NF-κB is a tran-scription factor that plays a key role in the regulation of genesassociated with inflammation, and its activation is one of thesignaling pathways through which Aβ exerts its neurotoxicity.5

The activated NF-κB regulates various pro-inflammatorymediators such as inducible nitric oxide synthase (iNOS),cyclooxygenase-2 (COX-2), and pro-inflammatory cytokinessuch as interleukins (ILs) and tumor necrosis factor α (TNF-α).6,7 When unstimulated, NF-κB exists mainly as inactiveheterodimers of p50/p65 in the cytoplasm binding with aninhibitory protein, IκB. On the other hand, in response tocellular stimulation, the IκB kinase complex is degraded and the

resulting free NF-κB then binds to κB binding sites in thepromoter regions of target genes that regulate transcription ofpro-inflammatory mediators.8 Activation of the NF-κB signalingpathway is intimately related with the activations of mitogen-activated protein kinase (MAPKs), which further stimulatedownstream transcription factors that promote pro-inflamma-tory gene expression.9 Characteristic MAPKs subfamilymembers are extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun NH2-protein kinase (JNK), and p38.10−12 Theseactivated MAPKs phosphorylate and activate other kinases ortranscription factors, consequently resulting in the alteration ofthe target gene expression.13

Corni fructus (CF), the fruit of Cornus officinalis Sieb. et Zucc,is a well-known food and medicinal herb in China, Japan, andKorea. CF has been shown to possess antidiabetic, anti-inflammatory, and antioxidative properties.14,15 In our previousstudy, p-coumaric acid (p-CA) and ursolic acid (UA) wereisolated from the ethyl acetate fraction of CF ethanol extract.We found that p-CA and UA specifically inhibited BACE116

and further suppressed Aβ25−35-induced reactive oxygen species(ROS) generation and apoptotic activity in PC12 cells.17 Toextend our knowledge of the neuroprotective effects of p-CAand UA, the anti-inflammatory activity of p-CA and UA againstAβ25−35-induced injury in PC12 cells was investigated and theirunderlying molecular mechanism of neuroprotective action waselucidated for the first time.

Received: March 17, 2014Revised: May 10, 2014Accepted: May 11, 2014Published: May 12, 2014

Article

pubs.acs.org/JAFC

© 2014 American Chemical Society 4911 dx.doi.org/10.1021/jf501314g | J. Agric. Food Chem. 2014, 62, 4911−4916

■ MATERIALS AND METHODSGeneral. Aβ25−35, p-CA, and UA were purchased from Sigma

Chemical Co. (St. Louis, MO, USA). RPMI 1640 medium, phosphate-buffered saline (PBS), fetal bovine serum, donor equine serum, trypsin0.25% solution, and penicillin/streptomycin solution were obtainedfrom Hyclone Laboratories, Inc. (Logan, UT, USA). N2 supplementand RPMI 1640 phenol red free medium were purchased Gibco BRL(Grand Island, NY, USA). Rabbit-antiphospho-iNOS, goat-antiphos-pho-COX-2, and goat-anti-β-actin were obtained from Santa CruzBiotechnology, Inc. (Santa Cruz, CA, USA). Rabbit antiphospho-JNK,rabbit-antiphospho-ERK, rabbit-antiphospho-IκB-α, and rabbit-anti-phospho-p65 monoclonal antibodies were purchased from CellSignaling Technology Inc. (Beverly, MA, USA). All other reagentsused in this study were of the highest grade available.Peptide Preparation and Cell Culture. Pheochromocytoma

PC12 cells (ATCC CRL 1721) have been extensively used as an invitro model system to study the mechanisms of neuronal toxicity.18,19

PC12 cells were purchased from ATCC (Manassas, VA, USA). PC12cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum at 37 °C in ahumidified atmosphere of 5% CO2 and 95% air. All cells were culturedin serum-free medium for 16 h prior to sample treatment. Sampleswere added at indicated concentrations 1 h prior to Aβ25−35 treatment,and the cells were plated at a density of 2 × 106 cells/well.The Aβ25−35 peptide used in this study preserves the toxicity of the

full length Aβ and is widely used to model Aβ-induced neuro-degeneration. The fragment Aβ25−35 has the central functional domainof the whole molecules Aβ that is required for neurotoxic effect.20,21

Aβ25−35 was dissolved in DMSO at a concentration of 10 mM andstored at −20 °C. The stock solution was diluted with PBS toappropriate concentration and incubated for 48 h at 37 °C foraggregation prior to each experiment. Samples were dissolved inDMSO and further diluted with PBS. The final concentration ofDMSO was adjusted to <0.01%, which was found to have no effect onthe cell viability in our preliminary experiments (data not shown).Western Blot Analysis. PC12 cells were incubated at 37 °C with

50 μM Aβ25−35 for 1 h for MAPKs family phosphorylation, for 4 h forNF-κB and I κB formation, and for 30 h for iNOS and COX-2activation with or without pretreatment with samples. The treated cellswere washed twice with cold PBS (pH 7.4) and harvested using a cellscraper. The cell pellets were resuspended in lysis buffer on ice for 1 h,and cell debris was removed by centrifugation at 13000 rpm for 10min. Protein concentrations were determined by the BCA proteinassay (Pierce, Rockford, IL, USA). Equal amounts of proteins weremixed with 4× Laemmli loading buffer and preheated at 95 °C for 5min. The samples were then loaded onto a 10% SDS−polyacrylamidegel and transferred onto a PVDF membrane for 1 h with a semidrytransfer system (Bio-Rad, Hercules, CA, USA). The membranes wereblocked with 5% nonfat milk in PBST with 0.1% Tween 20 for 1 h atroom temperature and then incubated overnight with anti-COX-2,anti-iNOS, antiphospho-p65, antiphospho-IκB-α, antiphospho-ERK1/2, antiphospho-JNK, and antiphospho-p38 antibodies diluted 1:1000in blocking solution at 4 °C. After five washes with PBS containingTween, the membrane was incubated with anti-goat or anti-rabbitimmunoglobulin G horseradish peroxidase-conjugated secondaryantibodies (diluted 1:1000) and visualized using Western blottingluminal reagents (Santa Cruz Biotechnology).Statistical Analysis. All experiments were performed in triplicate.

Data of each experiment showed the mean ± SD (n = 3). The valueswere compared with the control using analysis of variance followed byunpaired Student’s t test.

■ RESULTS AND DISCUSSION

Protective Effect of p-CA and UA on ProteinExpression of iNOS and COX-2 in Aβ25−35-InducedPC12 Cells. The chemical structures of p-CA and UA areshown in Figure 1. Our previous study demonstrated that p-CAand UA exhibited predominant neuroprotective effects against

Aβ25−35-induced injury via attenuating cellular oxidative stress,suppressing capsase-3 activity, and thus inhibiting apoptosis.17

Unlike p-CA, UA displayed adverse effects on cell viability atthe higher concentration, and treatment of high concentrationof UA showed neither protection nor exacerbation againstAβ25−35-induced damage.17 Thus, the experimental concen-tration limit of UA in the current study was adjusted to 20 μM,which did not affect cell viability. Concentration of UA (1, 10,and 20 μM) and p-CA (5, 25, and 50 μM) alone did not affectcell viability (data not shown). The concentration of 50 μMwas used for determining Aβ25−35-induced cell damage in thepresent experiments on the basis of our previous study.17 Todetermine whether p-CA and UA can suppress inflammation,the inflammatory responses were induced in PC12 cells byAβ25−35. COX-2 and iNOS are well-known pro-inflammatoryenzymes that play crucial roles in inflammatory responses.22 Asshown in Figure 2, Aβ25−35 significantly increased theexpression of COX-2 and iNOS (p < 0.01 and p < 0.001,respectively) compared with the control without Aβ25−35treatment. Cells pretreated with p-CA at 5 and 25 μM showedsignificantly reduced level of COX-2 expression in comparisonto the Aβ25−35-treated cells (p < 0.05). On the other hand,pretreatment of p-CA at 50 μM showed a somewhat weakereffect in COX-2 expression, which exerted neither positive nornegative effect against Aβ25−35-induced inflammation. Eachpretreatment of p-CA at 5, 25, and 50 μM strongly inhibitediNOS expression in a dose-dependent manner (p < 0.01). Evenat 5 μM p-CA inhibited Aβ25−35-induced iNOS expression. UAdose-dependently suppressed both iNOS and COX-2 levels.Even at 20 μM UA inhibited not only COX-2 expression butalso iNOS expression to a level much lower than the control.

Protective Effect of p-CA and UA on Aβ25−35-InducedNuclear Translocation of NF-κB Subunit p65 andPhosphorylation of IκB in PC12 Cells. When bindingwith IκB, NF-κB is inactive in the cytosol. However, it becomesactive via phosphorylation and degradation of IκB, andsubsequent nuclear translocation of NF-κB is stimulated byAβ.23 NF-κB signaling regulates the expression of downstreamNF-κB dependent gene products such as COX-2 and iNOS.24

Because the expression levels of COX-2 and iNOS enzymeswere reduced by p-CA and UA treatments, we further examinedwhether p-CA and UA could inhibit the nuclear translocation ofNF-κB subunit p65 by using Western blot. It has been reportedthat phosphorylation of p65 affects the transcriptional activityof NF-κB.25 As illustrated in Figure 3, the p-p65/p-IκB and β-actin ratios were significantly elevated by Aβ25−35 in PC12 cells(p < 0.05). UA exhibited significant and dose-dependentinhibition of p65 phosphorylation in comparison to theAβ25−35-stimulated control group (p < 0.05). p-CA alsoattenuated levels of p-p65 protein, however, somewhat lowerthan that of UA.

Figure 1. Chemical structures of p-coumaric acid and ursolic acid.

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Protective Effect of p-CA and UA on MAPKsPhosphorylation in Aβ25−35-Induced PC12 Cells. Becausethe activation of MAPKs family including ERK, JNK, and p38 isassociated with the NF-κB activation and downstreaminflammatory gene expression, we further investigated theactivation of MAPKs.26 The phosphorylation of p38, ERK, andJNK was significantly elevated when cells were treated withAβ25−35 alone compared with the control (Figure 4). p-CAsignificantly suppressed ERK1/2 and JNK phosphorylation butrelatively not that of p38 in response to Aβ25−35 treatment ofPC12 cells. In particular, p-CA at a concentration of 25 μMstrongly suppressed the phosphorylation of ERK1/2 and JNKeven to levels much lower than that of the control. UAexclusively decreased the phosphorylation of p-38, JNK, andERK in dose-dependent manners. In particular, the phosphor-ylation of ERK was markedly suppressed almost to its basallevel when treated with 1 μM UA. In addition, 20 μM UAsignificantly reduced the phosphorylation of p38 and JNK evento a level much lower than the control. p-CA and UA

comprehensively inhibited NF-κB activity, but the results of theupstream mechanisms were different.A deleterious effect of Aβ against neuronal cells has been

proved to be a critical cause of AD.27 Aβ accumulation occurs atan early step in the AD cascade of events in cellular dysfunctionthrough neuroinflammation.28 The discovery of NF-κB as a keytranscription factor in the inflammation provided a startingpoint that the suppression of NF-κB can be a promisingtherapeutic approach in inflammatory pathology such asarthritis, asthma, AD, and cancers.29 Various researchers haveproved that the activation of NF-κB is initiated by variousstimuli including Aβ, ROS, and inflammatory cytokines.23

Bioactive natural products have received much attention aspredominant and promising sources in the development ofnovel therapeutic agents. Synthetic agents have not replacednatural products as essential substances, and many of therecently developed medicines are derived from natural sources.In addition, therapeutic properties of various natural substanceswith anti-inflammatory effects may result from their inhibitionof NF-κB-dependent inflammation.30

Figure 2. Effects of p-coumaric acid (p-CA) and ursolic acid (UA) onAβ25−35-induced iNOS and COX-2 expression in PC12 cells. The totallysates of the proteins were subjected to Western blot analysis, asdescribed under Materials and Methods. Cells were pretreated withsamples at the indicated concentrations for 1 h and then exposed to 50μM Aβ25−35 for 30 h. The ratio of immunointensity between COX-2/iNOS and β-actin was calculated. Each bar represents the mean ± SDfrom three independent experiments. (###) p < 0.001 and (##) p <0.01 versus control group. (∗∗∗) p < 0.001, (∗∗) p < 0.01, and (∗) p <0.05 versus the group treated with Aβ25−35 alone.

Figure 3. Effects of p-coumaric acid (p-CA) and ursolic acid (UA)against Aβ25−35-induced phosphorylation of NF-κB subunit (p65) anddegradation of IκB in PC12 cells. The total lysates of the proteins weresubjected to Western blot analysis, as described under Materials andMethods. Cells were pretreated with the indicated concentrations ofsamples for 1 h and then exposed to 50 μM Aβ25−35 for 4 h. The ratioof immunointensity between the p-p65/IκB-α and β-actin wascalculated. Each bar represents the mean ± SD from threeindependent experiments. (#) p < 0.05 versus control group. (∗∗) p< 0.01 and (∗) p < 0.05 versus the group treated with Aβ25−35 alone.

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p-CA [3-(4-hydroxyphenyl)-2-propenoic acid], a ubiquitousintermediate metabolite in the synthesis of many polyphenolsincluding flavonoids in plants, is widely found in fruits,vegetables, and cereals.31 Previous investigators have shownthat p-CA protected dextran sodium sulfate induced-intestinalinflammation through the suppression of COX-2 expression.32

Apart from this, no report regarding the anti-inflammatoryproperty of p-CA via suppression of the NF-κB pathway hasbeen found so far.

UA (3β-hydroxyurs-12-en-28-oic acid) is a pentacyclictriterpene acid widely distributed in berries, flowers, and fruits.Previous research has demonstrated that UA inhibited LPS-induced inflammatory response through inhibition of NF-κBand iNOS expression in lung epithelial cells.33 In addition,animal studies have shown that oral administration of UA (10mg/kg·day) attenuated D-galactose-induced inflammatory re-sponse in the mouse prefrontal cortex through down-regulationof iNOS and COX-2 expression.34 Another study hasdemonstrated that UA enhances collateral blood flow recoverythrough induction of neovascularization in a hind limb ischemiamouse model.35

The present study suggested for the first time that p-CA andUA isolated from CF efficiently suppressed the expression ofiNOS and COX-2 in Aβ25−35-treated PC12 cells. Theseinhibitions appeared to correlate with the inhibition of NF-κBactivation as pretreating cells with p-CA and UA blocked thephosphorylation of the inhibitory subunit IκB. Similarinhibitory effects of UA on iNOS and COX-2 regulated byNF-κB inactivation have been observed in several different celltypes including macrophages, platelets, and leukemic andcancer cells exposed to diverse oxidative or inflammatorystimuli.36−38 Our results show that p-CA might exert its anti-inflammatory ability through inhibiting p38 and JNK pathwaysleading to NF-κB inactivation and thereby suppressingexpressions of inflammatory target proteins including COX-2and iNOS. On the other hand, UA may affect pro-inflammatorysignaling through the inhibition of ERK, p38, and JNK, leadingto NF-κB inactivation and thereby suppressing expressions ofinflammatory target proteins including COX-2 and iNOS. Yehet al. recently reported that UA suppressed migration of humanbreast cancer cells by reducing the level of JNK phosphor-ylation through inhibition of NF-κB.39 For any compounds toexert direct neuroprotective actions they have to permeate theblood−brain barrier (BBB). UA has previously been found inthe brain, which implied UA can cross the BBB.40 Furtherstudies regarding the BBB penetration by p-CA are needed.Our novel findings suggest that p-CA and UA have potentialneuroprotective effects against Aβ25−35-induced neurotoxicitythrough blocking iNOS and COX-2 via down-regulation of NF-κB and MAPKs pathways.The results indicate that p-CA and UA derived from CF

suppressed Aβ25−35-stimulated expression of pro-inflammatorymediators such as iNOS and COX-2 in PC12 cells.Furthermore, p-CA and UA enhanced the inhibitory effectsagainst inflammation via inactivation of both NF-κB andMAPKs signaling pathways. Even though PC12 cells displaydistinctive sensitivity to neurotoxicity and have been extensivelyused in the studies of Aβ-induced injury, they cannotthoroughly represent the primary cultured neurons. Therefore,further studies are required to concentrate on primary neuronsand in vivo models. Taken together, these findings are the firstto represent the anti-inflammatory effects of p-CA and UAagainst Aβ25−35 toxicity in PC12 cells, which may provide thepharmacological basis underlying the prevention of AD.

■ AUTHOR INFORMATION

Corresponding Author*(M.J.) Phone: +82-51-200-7323. Fax: +82-51-200-7535. E-mail: mjun@dau.ac.kr.

Author Contributions∥J.-H.Y. and K.Y. contributed equally to this work.

Figure 4. Effects of p-coumaric acid (p-CA) and ursolic acid (UA)against Aβ25−35-induced phosphorylation of MAPKs in PC12 cells.The total lysates of the proteins were subjected to Western blotanalysis, as described under Materials and Methods. Cells werepretreated with samples for 1 h and then exposed to 50 μM Aβ25−35 for1 h. The ratio of immunointensity between the p-p38/ERK1/2/JNKand β-actin was calculated. Each bar represents the mean ± SD fromthree independent experiments. (∗∗) p < 0.01 and (#) p < 0.05 versuscontrol group. (∗∗) p < 0.01 and (∗) p < 0.05 versus the group treatedwith Aβ25−35 alone.

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FundingThis research was supported by the Basic Science ResearchProgram through the National Research Foundation (NRF)funded by the Ministry of Education, Science and Technology(2012-006723). This research was also partially supported bythe Brain Busan 21 project in 2013.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe are grateful to Y. Hwang for isolating and providing p-coumaric acid and ursolic acid from Corni f ructus.

■ ABBREVIATIONS USEDAD, Alzheimer’s disease; Aβ, β-amyloid; BACE1, β-secretase;NF-κB, nuclear factor κB; iNOS, inducible nitric oxidesynthase; COX-2, cyclooxygenase-2; ILs, interleukins; TNF-α,tumor necrosis factor α; MAPKs, mitogen-activated proteinkinase; ERK 1/2, extracellular signal regulated kinase 1/2; JNK,c-Jun NH2-protein kinase; CF, Corni f ructus; p-CA, p-coumaricacid; UA, ursolic acid; PBS, phosphate-buffered saline; PC12cell, pheochromocytoma cell

■ REFERENCES(1) Selkoe, D. J. Translating cell biology into therapeutic advances inAlzheimer’s disease. Nature 1999, 399, A23−A31.(2) Hardy, J. Amyloid the presenilins and Alzheimer’s disease. TrendsNeurosci. 1997, 20, 154−159.(3) Szaingurten-Solodkin, I.; Hadad, N.; Levy, R. Regulatory role ofcytosolic phospholipase A2α in NADPH oxidase activity and ininducible nitric oxide synthase induction by aggregated Aβ1-42 inmicroglia. Glia 2009, 57, 1727−1740.(4) In’t Veld, B. A.; Ruitenberg, A.; Hofman, A.; Launer, L. J.; VanDuijn, C. M.; Stijnen, T.; Breteler, M. M. B.; Stricker, B. H. C.Nonsteroidal anti-inflammatory drugs and the risk of Alzheimer’sdisease. N. Engl. J. Med. 2001, 22, 1515−1521.(5) Kaltschmidt, B.; Uherek, M.; Volk, B.; Baeuerie, P. A.;Kaltschmidt, C. Transcription factor NF-κB is activated in primaryneurons by amyloid β peptides and in neurons surrounding earlyplaques from patients with Alzheimer’s disease. Proc. Natl. Acad. Sci.U.S.A. 1997, 94, 2642−2647.(6) Yun, K. J.; Koh, D. J.; Kim, S. H.; Park, S. J.; Ryu, J. H.; Kim, D.G.; Lee, J. Y.; Lee, K. T. Anti-inflammatory effects of sinapic acidthrough the suppression of inducible nitric oxide synthase, cyclo-oxygase-2, and proinflammatory cytokines expressions via nuclearfactor-κB inactivation. J. Agric. Food Chem. 2008, 56, 10265−10272.(7) Tsai, S. J.; Lin, C. Y.; Mong, M. C.; Ho, M. W.; Yin, M. C. S-Ethylcysteine and S-propyl cysteine alleviate β-amyloid induced cytotoxicityin nerve growth factor differentiated PC12 cells. J. Agric. Food Chem.2010, 58, 7104−7108.(8) Nolan, G. P.; Ghosh, S.; Liou, H. C.; Tempst, P.; Baltimore, D.DNA binding and IκB inhibition of the cloned p65 subunit of NF-κB,a rel-related polypeptide. Cell 1991, 64, 961−969.(9) Saklatvala, J. Inflammatory signaling in cartilage: MAPK and NF-κB pathways in chondrocytes and the use of inhibitors for researchinto pathogenesis and therapy of osteoarthritis. Drug Targets 2007, 8,305−313.(10) Davis, R. The mitogen-activated protein kinase signaltransduction pathway. J. Biol. Chem. 1993, 268, 14553−14556.(11) Su, B.; Karin, M. Mitogen-activated protein kinase cascades andregulation of gene expression. Curr. Opin. Immunol. 1996, 8, 402−411.(12) Ichijo, H. From receptors to stress-activated MAP kinases.Oncogene 1999, 18, 6087−6093.(13) Hwang, D.; Jang, B. C.; Yu, G.; Boudreau, M. Expression ofmitogen-inducible cyclooxygenase induced by lipopolysaccharide.Biochem. Pharmacol. 1997, 54, 87−96.

(14) Gao, D.; Li, N.; Li, Q.; Li, J.; Han, Z.; Fan, Y.; Liu, Z. Study ofthe extraction, purification and antidiabetic potential of ursolic acidfrom Cornus of f icinalis Sieb. et Zucc. Therapy 2008, 5, 697−705.(15) Park, C. H.; Noh, J. S.; Tanaka, T.; Yokozawa, T. Effects ofmorroniside isolated from Corni f ructus on renal lipids andinflammation in type 2 diabetic mice. J. Pharm. Pharmacol. 2010, 62,374−380.(16) Youn, K.; Jun, M. Effects of key compounds isolated from Cornif ructus on BACE1 inhibition. Phytother. Res. 2012, 26, 1714−1718.(17) Hong, S. Y.; Jeong, W. S.; Jun, M. Protective effects of the keycompounds isolated from Corni f ructus against β-amyloid-inducedneurotoxicity in PC12 cells. Molecules 2012, 17, 10831−10845.(18) Jang, J. H.; Surh, Y. J. Protective effects of resveratrol onhydrogen peroxide-induced apoptosis in rat pheochromocytoma(PC12) cells. Mutat. Res. 2001, 496, 181−190.(19) Zhu, J. T.; Choi, R. C.; Chu, G. K.; Cheung, A. W.; Gao, Q. T.;Li, J.; Jiang, Z. Y.; Dong, T. T.; Tsim, K. W. Flavonoids possessneuroprotective effects on cultured pheochromocytoma PC12 cells: acomparison of different flavonoids in activating estrogenic effect and inpreventing β-amyloid-induced cell death. J. Agric. Food. Chem. 2007,55, 2438−2445.(20) Qu, M.; Li, L.; Chen, C.; Li, M.; Pei, L.; Chu, F.; Yang, J.; Yu, Z.;Wang, D.; Zhou, Z. Protective effects of lycopene against amyloid β-induced neurotoxicity in cultured rat cortical neurons. Neurosci. Lett.2011, 505, 286−290.(21) Luo, S.; Lan, T.; Liao, W.; Zhao, M.; Yang, H. Genistein inhibitsAβ25−35-induced neurotoxicity in PC12 cells via PKC signalingpathway. Neurochem. Res. 2012, 37, 2787−2794.(22) Zhang, X.; Cao, J.; Zhong, L. Hydroxytyrosol inhibits pro-inflammatory cytokines, iNOS, and COX-2 expression in humanmonocytic cells. Naunyn. Schmiedebergs Arch. Pharmacol. 2009, 379,581−586.(23) Mattson, M. P.; Camandola, S. NFκB in neuronal plasticity andneurodegenerative disorders. J. Clin. Invest. 2001, 107, 247−254.(24) Fang, M.; Wang, J.; Han, S.; Hu, Z.; Zhan, J.; Ling, S.; Rudd, J.A.; Geng, Y. Protective effects of ω-conotoxin on amyloid-β-induceddamage in PC12 cells. Toxicol. Lett. 2011, 206, 325−338.(25) Lentsch, A. B.; Ward, P. A. The NFκB/IκB system in acuteinflammation. Arch. Immunol. Ther. Exp. (Warsz.) 2000, 48, 59−63.(26) Lee, S. Y.; Lee, J. W.; Lee, H.; Yoo, H. S.; Yun, Y. P.; Oh, K. W.;Ha, T. Y.; Hong, J. T. Inhibitory effect of green tea extract on β-amyloid-induced PC12 cell death by inhibition of the activation ofNFκB and ERK/p38 MAP kinase pathway through antioxidantmechanisms. Mol. Brain Res. 2005, 140, 45−54.(27) Yan, S. D.; Zhu, H.; Fu, J.; Yan, S. F.; Roher, A.; Tourtellotte, W.W.; Rajavashisth, T.; Chen, X.; Godman, G. C.; Stern, D.; Schmidt, A.M. Amyloid-β peptide-receptor for advanced glycation end productinteraction elicits neuronal expression of macrophage-colony stimulat-ing factor: a proinflammatory pathway in Alzheimer’s disease. Proc.Natl. Acad. Sci. U.S.A. 1997, 94, 5296−5301.(28) Rosales-Corral, S.; Tan, D. X.; Reiter, R. J.; Valdivia-Velazquez,M.; Acosta-Martínez, J. P.; Ortiz, G. G. Kinetics of the neuro-inflammation-oxidative stress correlation in rat brain following theinjection of fibrillar amyloid-β onto the hippocampus in vivo. J.Neuroimmunol. 2004, 150, 20−28.(29) Chen, F.; Castranova, V.; Shi, X.; Demers, L. M. New insightsinto the role of nuclear factor-κB, aubiquitous transcription factor inthe initiation of diseases. Clin. Chem. 1999, 45, 7−17.(30) Heras, B.; Hortelano, S. Molecular basis of the anti-inflammatory effects of terpenoids. Inflamm. Allergy Drug Targets2009, 8, 28−39.(31) Luceri, C.; Giannini, L.; Lodovici, M.; Antonucci, E.; Abbate, R.p-Coumaric acid, a common dietary phenol, inhibits platelet activity invitro and in vivo. Br. J. Nutr. 2007, 97, 458−463.(32) Luceri, C.; Guglielmi, F.; Lodovici, M.; Giannini, L.; Messerini,L.; Dolara, P. Plant phenolic 4-coumaric acid protects against intestinalinflammation in rats. Scand. J. Gastroenterol. 2004, 39, 1128−1133.(33) Lee, C. H.; Wu, S. L.; Chen, J. C.; Li, C. C.; Lo, H. Y.; Cheng,W. Y.; Lin, J. G.; Chang, Y. H.; Hsiang, C. Y.; Ho, T. Y. Eriobotrya

Journal of Agricultural and Food Chemistry Article

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japonica leaf and its triterpenes inhibited lipopolysaccharide-inducedcytokines and inducible enzyme production via the nuclear factor-κBsignaling pathway in lung epithelial cells. Am. J. Chin. Med. 2008, 36,1185−1198.(34) Lu, J.; Wu, D.; Zheng, Y.; Hu, B.; Zhang, Z.; Ye, Q.; Liu, C.;Shan, Q.; Wang, Y. Ursolic acid attenuates D-galactose-inducedinflammatory response in mouse prefrontal cortex through inhibitingAGEs/RAGE/ NFκB pathway activation. Cereb. Cortex 2010, 20,2540−2548.(35) Lee, A.; Chen, T. L.; Shih, C. M.; Huang, C. Y.; Tsao, N. W.;Chang, N. C.; Chen, Y. H.; Fong, T. H.; Lin, F. Y. Ursolic acid inducesallograft inflammatory factor-1 expression via a nitric oxide-relatedmechanism and increases neovascularization. J. Agric. Food Chem.2010, 58 (24), 12941−12949.(36) Najid, A.; Simon, A.; Cook, J.; Chable-Robinovitch, H.; Delage,C.; Chulia, A. J.; Rugaud, M. Characterization of ursolic acid as alipoxygenase and cyclooxygenase inhibitor using macrophages,platelets and differentiated HL60 leukemic cells. FEBS Lett. 1992,299, 213−217.(37) Suh, N.; Honda, T.; Finlay, H. J.; Barchowsky, A.; Williams, C.;Benoit, N. E.; Xie, Q. W.; Nathan, C.; Gribble, G. W.; Sporn, M. B.Novel triterpenoids suppress inducible nitric oxide synthase (iNOS)and inducible cyclooxygenase (COX-2) in mouse macrophages. CancerRes. 1998, 58, 717−723.(38) Li, Y.; Xing, D.; Chen, Q.; Chen, W. R. Enhancement ofchemotherapeutic agent-induced apoptosis by inhibition of NFκBusing ursolic acid. Int. J. Cancer 2009, 127, 462−473.(39) Yeh, C. T.; Wu, C. H.; Yen, G. C. Ursolic acid, a naturallyoccurring triterpenoid, suppresses migration and invasion of humanbreast cancer cells by modulating c-Jun N-terminal kinase, Akt andmammalian target of rapamycin signaling. Mol. Nutr. Food Res. 2010,54, 1285−1295.(40) Chen, Q.; Luo, S.; Zhang, Y. Development of a liquidchromatography-mass spectrometry method for the determination ofursolic acid in rat plasma and tissue: application to thepharmacokinetic and tissue distribution study. Anal. Bioanal. Chem.2011, 399, 2877−2884.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf501314g | J. Agric. Food Chem. 2014, 62, 4911−49164916