This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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2 Food Chemistry 128 (11) Contents lists available at SieneDiret Food Chemistry journal homepage: Flavonoids from aai (Euterpe oleraea Mart.) pulp and their antioxidant and anti-inflammatory ativities q Jie Kang a, Chenghui Xie a, Zhimin Li b, Shanmugam Nagarajan a, Alexander G. Shauss, Tong Wu b,, Xianli Wu a, a USDA Arkansas Children s Nutrition Centre, Department of Physiology and Biophysis, University of Arkansas for Medial Sienes, 15 Children s Way, Little Rok, AR 722, USA b Shanghai Institute of Pharmaeutial Industry, 13 W. Beijing Road, Shanghai, China AIBMR Life Siene In., 4117 S Meridian, Puyallup, WA 98373, USA artile info abstrat Artile history: Reeived 6 January 11 Reeived in revised form 31 January 11 Aepted 1 Marh 11 Available online 5 Marh 11 Keywords: Aai Euterpe oleraea Mart. Flavonoid Antioxidant Anti-inflammation Five flavonoids, (2S,3S)-dihyrokaempferol 3--b-D-gluoside (1) and its isomer (2R,3R)-dihydrokaempferol 3--b-D-gluoside (2), isovitexin (3), velutin (4) and 5,4 -dihydroxy-7,3,5 -trimethoxyflavone (5), were isolated from aai (Euterpe oleraea Mart.) pulp. The strutures of these ompounds were eluidated based upon spetrosopi and hemial analyses. To our knowledge, ompounds 1, 2, 4 and 5 were identified from aai pulp for the first time. The in vitro antioxidant ativities of these ompounds were evaluated by the oxygen radial absorbane apaity (RAC) assay. The RAC values varied distintly ( lmol Trolox equivalent (TE)/g) from 5,4 -dihydroxy-7,3,5 -trimethoxyflavone (5) to isovitexin (3) and were affeted by the numbers/positions of hydroxyl groups, substitute groups, as well as stereo onfiguration. The anti-inflammatory effets of these ompounds were sreened by the sereted embryoni alkaline phosphatase (SEAP) reporter assay, whih is designed to measure NF-jB ativation. Velutin (4) was found to dose-dependently inhibit SEAP seretion in RAW-blue ells indued by LPS, with an IC 5 value of 2. lm. Velutin (4) also inhibited SEAP seretion indued by oxidised LDL, indiating potential athero-protetive effets. Ó 11 Elsevier Ltd. All rights reserved. 1. Introdution Aai fruits gained popularity in North Ameria and in the European ountries lately as a new super fruit largely due to its extremely high antioxidant apaity and potential anti-inflammatory ativities (Shauss, Wu, Prior, u, Huang, et al., 6). Antioxidant and anti-inflammatory ativities of aai pulp or aai juie have been studied in human, animal and ell ulture models (Del Pozo-Insfran, Perival, & Talott, 6; Jensen et al., 8; Spada et al., 9). In a reent study of the effets of aai pulp supplementation on the lifespan of oxidative stressed female flies, moleular analysis revealed that supplementation with aai pulp restored the transript level of lethal (2) essential for life (l(2)efl), a downstream event of the oxidative stress response pathway Jun-N-terminal kinase (JNK), in loss-of-funtion and redution-of-funtion sod1mutants. This resulted in a signifiant life span extension, suggesting q Mentioning of trade names or ommerial produts in this publiation is solely for the purpose of providing speifi information and does not imply reommendation or endorsement by the US Department of Agriulture. Corresponding authors. Tel.: (T. Wu), tel.: ; fax: (X. Wu). addresses: tongwu88@163.om (T. Wu), wuxianli@uams.edu (X. Wu). that the ompounds in aai may play a role in ell signalling and hanges in gene expression (Sun et al., 1). Major polyphenoli omponents in aai pulp inlude anthoyanins, proanthoyanidins, other flavonoids and lignans (Chin, Chai, Keller, & Kinghorn, 8; Gallori, Bilia, Bergonzi, Barbosa, & Vinieri, 4; Shauss, Wu, Prior, u, Patel, et al., 6). Among them, the flavonoids were found to be the major polyphenols. Flavonoids are ubiquitously present in fruits and vegetables. As a group, flavonoids have been shown to exhibit strong antioxidant apaities. The mehanism of antioxidant ativity of flavonoids involves the diret savenging or quenhing of oxygen free radials or exited oxygen speies, as well as the inhibition of oxidative enzymes that generate these reative oxygen speies (Pietta, ; Terao, 9). Flavonoids have also shown anti-inflammatory ativity in both the proliferative and exudative phases of inflammation (Rathee et al., 9). In our previous paper (Kang et al., 1), seven known flavonoids were isolated and their antioxidant ativities were evaluated using three assays. The first objetive of this study was to isolate and identify additional flavonoids from aai pulp. Furthermore, antioxidant and anti-inflammatory ativities were evaluated by the oxygen radial absorbane apaity (RAC) assay and the sereted embryoni alkaline phosphatase (SEAP) reporter assay /$ - see front matter Ó 11 Elsevier Ltd. All rights reserved. doi:1.116/j.foodhem

3 J. Kang et al. / Food Chemistry 128 (11) The SEAP reporter assay was designed to measure the nulear fator-kappa B (NF-jB) ativation (Berger, Hauber, Hauber, Geiger, & Cullen, 1988; Moon, Hahn, Lee, & Kim, 1). As a major transription fator, NF-jB plays a key role in regulating the genes responsible for innate and adaptive immune responses (Brasier, 6; Hoffmann, Natoli, & Ghosh, 6). The ativation of NF-jB has been shown to mediate inflammation by inreasing the expression of pro-inflammatory ytokines, hemokines and enzymes (Pahl, 1999). In the SEAP reporter assay, lipopolysaharide (LPS) or oxidised LDL (oxldl) was used as stimuli; the later one may partiularly impliate potential protetive ations of test ompounds against atheroslerosis. 2. Materials and methods 2.1. Instrumentation ptial rotations were measured on a Perkin-Elmer 341 digital polarimeter (Waltham, USA). The UV spetra were obtained on a Shimadzu-25PC spetrophotometer (Kyoto, Japan). The irular dihroism (CD) spetra were reorded in Me using a JASC J- 81 spetropolarimeter (Easton, USA). The infrared (IR) spetra were obtained with a NEXUS 67-FTIR spetrophotometer (San Jose, USA). 1 H and 13 C NMR spetra were reorded on a Varian Inova 5 MHz NMR spetrometer (Palo Alto, USA). Eletrospray ionisation mass spetrometry (ESIMS) was arried out using Miromass Q-Tof mass spetrometer (Milford, USA). High performane liquid hromatography (HPLC) was performed in a Agilent 1 HPLC equipped with a UV detetor (Palo Alto, USA); a C-18 Dikma Diamonsil analytial olumn ( mm, 5 lm) (Sarborough, Canada) and a C-18 Waters ubondapak olumn (3 7.8 mm, 1 lm) (Milford, USA) were employed for analysis and preparation respetively Plant material The fruits of Euterpe oleraea Mart. were harvested in Para state, Brazil, in September, 9. A vouher speimen was deposited at Embrapa Amazônia riental (Belém, Brazil). The fruits were proessed within hours of harvesting to pure pulp and stored at C until transferred for freeze drying. The frozen pulp was lyophilised at Liotenia Tenologia em Alimentos (Sao Paolo, Brazil) and the freeze-dried pulps were transported into the US and supplied by Earth Fruits (South Jordan, USA) Chemials and reagents Methanol (Me), 95% ethanol (Et), n-butyl alohol (n- Bu), petrol ether, and hloroform (CHCl 3 ) were purhased from Shanghai Zhengxing Chemial (Shanghai, China). Ethyl aetate (EtA) and aetone were obtained from Sinopharm Chemial Reagent (Shanghai, China). Silia gel ( mesh) and Sephadex LH- were supplied by the Branh of Qingdao Marine Chemial (Qingdao, China) and Shanghai Juyuan Biotehnology (Shanghai, China), respetively. Diatomite was obtained from Sinopharm Chemial Reagent (Shanghai, China). 2,2 -Azobis(2-amidinopropane)dihydrohloride (AAPH) was purhased from Wako Chemials USA (Rihmond, USA). 6-Hydroxy-2,5,7,8-tetramethylhroman-2-arboxyli aid (Trolox) and fluoresein (sodium salt) (FL) were obtained from Aldrih (Milwaukee, USA). Potassium phosphate dibasi (K 2 HP 4 ), potassium phosphate monobasi (KH 2 P 4 ) and Miroplates (48-well, Falon 323) were obtained from VWR (West Chester, USA) Extration and isolation proedure The freeze-dried aai pulp powder (3 g) was mixed with diatomite, perolated with 95% alohol for two weeks. After evaporation of the solvents under the vauum, the residue (5 g) was then extrated with petroleum ether, CHCl 3, EtAC, and n-bu, in suession. The EtA and n-bu frations were further separated by open olumn with silia gel, Sephadex LH-, preparative thin layer hromatography (PTLC) and preparative high performane liquid hromatography (HPLC) respetively. The EtAC extrat (5 g) were loaded into an open silia gel olumn and eluted with CHCl 3 Me mixtures with inreasing polarity. Fration (CHCl 3 :Me = 9:1) was subjeted to a Sephadex LH- olumn, eluted with Me. Sub-fration was then purified by PTLC (petroleum ether:eta = 1:3) to get ompounds 4 (R f =.75, 4 mg) and 5 (R f =.81, 3 mg). Fration (CHCl 3 :Me = 1:1) was submitted to a Sephadex LH- olumn and eluted with Me. Sub-fration was further purified using HPLC with a Waters ubondapak olumn. The mobile phase was CH 3 CN H 2 (ontaining.1% formi aid) (:, v/v), at a flow rate of 1 ml/min to yield ompound 3 (3 mg). The hromatographi profile was deteted at 27 nm.n- Bu extrat ( g) was separated with a Sephadex LH- olumn eluted with Me, followed by further purifiation using HPLC on a Waters ubondapak olumn (flow rate, 1 ml/min; detetion wave length, 27 nm), with CH 3 CN H 2 (ontaining.1% formi aid) (3:7, v/v) as the mobile phase, to yield ompounds 1 (15 mg) and 2 (7 mg) RAC assay The RAC assay was onduted based on the method reported previously (Wu et al., 4). Briefly, the assay was arried out on a FLUstar Galaxy plate reader (BMG Labteh, Durham, USA) used with fluoresene filters for an exitation wavelength of 485 nm and an emission wavelength of 5 nm. The temperature of the inubator was set to 37 C. Fluoresein was used as fluoresene probe; 2,2 -azobis (2-methylpropionamide) dihydrohloride (AAPH) was used as peroxyl generator; Trolox was used as standard. The results were expressed as lmol TE per gram SEAP reporter assay RAW-Blue ells (Invitrogen, San Diego, USA) are derived from RAW264.7 marophages with hromosomal integration of a SEAP reporter onstrut induible by NF-jB and AP-1. RAW-Blue mouse marophage ell lines were ultured in Dulbeo s Modified Eagle s Medium (DMEM) supplemented with 1% (v/v) fetal bovine serum (FBS) (Hylone, Logan, USA) and zeoineosin ( lg/ml). All ell ulture reagents were purhased from Invitrogen (San Diego, USA). RAW-Blue ells (1 1 5 ells/well) were pretreated with ompounds for 3 h, stimulated by LPS ( ng/ml, Invitrogen, San Diego, USA) or oxldl ( ng/ml, Aademy Biomedial, Houston, USA) for 18 h. Luteolin was isolated from the aai pulp desribed in our previous study (Kang et al., 1). The supernatants were olleted for the SEAP seretion assay. The QUANTI-Blue powder was dissolved in endotoxin-free water and sterile filtered (.22 lm) (QuantiQuanta-blue substrate). RAW-Blue ell supernatant ( ll/well) was added to QuantiQuanta-blue substrate (1 ll/ well) and inubated at 37 C for.5 1 h. The absorbane was measured at 6 nm in a Polarstar miroplate reader (BMG Labteh, Durham, USA).

4 154 J. Kang et al. / Food Chemistry 128 (11) Statistial analysis The results for anti-inflammatory assays were expressed as mean ± SD (n = 3). Data were subjeted to one-way ANVA for statistial analyses and the Student Newman Keuls Method for multiple omparison proedures. A value of P <.5 was onsidered as signifiant differene. Statistial analyses were performed using SigmaStat statistial software (SigmaStat 3.5). 3. Results and disussion 3.1. Identifiation of isolated ompounds Five flavonoids were obtained (Fig. 1) from aai (Euterpe oleraea Mart.) pulp. Their strutures were eluidated by ESIMS, UV, IR, 1 H and 13 C NMR, CD spetra and by omparison with the literature. (2S,3S)-dihyrokaempferol 3--b-D-gluoside (1): yellow amorphous powder; ½aŠ 25 D (.2, Et); UV (Me) max 292, 3 (sh) nm; IR (KBr) v max 35 (), 2919 (CH), 1642 (C@), 1164, 178 (C ) m 1 ; ESIMS m/z 473 [M + Na] + ; for the 1 H and 13 C NMR data see Table 1 and for CD spetral data see Table 2. The data were onsistent with the known ompound (Kato, Li, Koike, Wang, & Koike, 1). (2R,3R)-dihydrokaempferol 3--b-D-gluoside (2): yellow amorphous powder; ½aŠ 25 D (.2, Et); UV (Me) max 292, 3 (sh) nm; IR (KBr) v max 35 (), 2919 (CH), 1642 (C@), 1164, 178 (C ) m 1 ; ESIMS m/z 473 [M + Na] + ; for 1 H and 13 C NMR data see Table 1 and for CD spetral data see Table 2. The data were onsistent with the known ompound (Yu, Li, Chen, & Yang, 1992). Isovitexin (3): yellowish amorphous powder; ESIMS: m/z = 431 [M H] ; 1 H NMR (DMS-d 6 ) ppm: d 13.5 (1H, s, 5-), 7.89 (2 H, d, J= 8.4 Hz, H-2, 6), 6.92 (2 H, d, J= 8.4 Hz, H-3, 5), 6.7 (1 H, s, H- 3), 6.44 (1 H, s, H-8), 4. (1 H, d, J= 1. Hz, H-glu-1). The data were onsistent with the known ompound (Leong et al., 1). Table 1 1 H and 13 C NMR spetral data (5 and 125 MHz, DMS-d 6 ) of ompounds 1 and 2. Position 1 (2S,3S) 2 (2R, 3R) d H (J in Hz) d C d H (J in Hz) d C Aglyone moiety (d, 7.) (d, 8.) (d, 7.) (d, 7.) (s) (s) (s) (s) a a (d, 8.) (d, 8.4) (d, 8.) (d, 8.4) (d, 8.) (d, 8.4) (d, 8.) (d, 8.4) 129. Gluose moiety 1 4. (d, 8.) (d, 6.4) (m) (m) (m) (5H, m) (m) (m) (m) (m) Velutin (4): yellowish amorphous powder; ESIMS: m/z = 313 [M H] ; 1 H NMR (DMS-d 6 ) ppm: d (1H, s, 5-), 7. (1 H, d, J= 8.5 Hz, H-6), 7.58 (1 H, brs, H-2), 6.94 (1 H, d, J= 8.5 Hz, H-5), 6.95 (1 H, s, H-3), 6. (1 H, d, J= 2. Hz, H-8), 6.37 (1 H, d, J= 2. Hz, H-6), 3.89 (3 H, s, CH 3-3), 3.87 (3 H, s, CH 3-7). The data were onsistent with the known ompound (Çitoğlu, Sever, Antus, Baitz-Gás, & Altanlar, 3).5,4 -Dihydroxy-7,3,5 -trimethoxyflavone (5): yellowish amorphous powder; ESIMS: m/ z = 343 [M H] ; 1 H NMR (DMS-d 6 ) ppm: d (1H, s, 5-), 8.34 (1 H, s, 4-), 7.37 (2 H, s, H-2, 6), 7.6 (1 H, s, H-3), 6.87 (1 H, brs, H-8), 6.38 (1 H, brs, H-6), 3.89 (6 H, s, CH 3-3, 5), 3.88 S 2 S 3 R R CH 3 H 3 C R gl 3 4 R=H 5 R=CH 3 Fig. 1. Chemial strutures of five flavonoids, (2S,3S)-dihyrokaempferol 3--b-D-gluoside (1), (2R,3R)-dihydrokaempferol 3--b-D-gluoside (2), isovitexin (3), velutin (4) and 5,4 -dihydroxy-7,3,5 -trimethoxyflavone (5) isolated from aai (Euterpe oleraea Mart.) pulp.

5 J. Kang et al. / Food Chemistry 128 (11) Table 2 CD spetra of ompounds 1 and 2. Compounds n?p n?p (2S,3S)-Dihydrokaempferol 3-b-D-gluoside [h] [h] (1) (2R,3R)-Dihydrokaempferol 3-b-Dgluoside (2) [h] [h] 29 27,329 (3 H, s, CH 3-7). The data were onsistent with the known ompound (Zahir et al., 1996). Among these five flavonoids, ompounds 1 and 2 were found to be isomers, aording to their spetrosopi data. Slight differenes in the spetra between 1 and 2 suggested that these ompounds have the same struture type but different stereo onfigurations. The most remarkable differene was the anomeri proton of gluopyranosyl residue (1: d H = 4., 2: d H = 3.97). The CD urve of the two ompounds showed Cotton effets at 324 and 29 nm (Table 2). The absolute onfigurations were eluidated to be (2S, 3S) for 1 and (2R, 3R) for 2 aording to the literatures (Sakushima, hno, Coskun, Seki, & hkura, 2; Yu et al., 1992). To our knowledge, (2S,3S)-dihyrokaempferol 3--b-D-gluoside (1) and its isomer (2R,3R)-dihydrokaempferol 3--b-D-gluoside (2), velutin (4) and 5,4 -dihydroxy-7,3,5 -trimethoxyflavone (5) were identified from aai pulp for the first time Antioxidant apaities of the flavonoids RAC is a hemial antioxidant assay that is based on the inhibition of the peroxyl-radial indued oxidation initiated by thermal deomposition of AAPH. It is one of the most widely used in vitro antioxidant apaity assays (Prior, Wu, & Shaih, 5). Similar to what we observed from the previous study (Kang et al., 1), the RAC values of these five ompounds varied distintly, from ± 8.8 lmol TE/g for 5,4 -dihydroxy-7,3,5 -trimethoxyflavone (5) to ± lmol TE/g for isovitexin (3) (Table 3). The numbers and positions of the hydroxyl groups and/or other substitutes, suh as through glyosylation have been shown to affet the antioxidant apaity of flavonoids (Cao, Sofi, & Prior, 1997; Kang et al., 1; Prasad, Divakar, Shivamurthy, & Aradhya, 5). Methylation of hydroxyl groups on benzene rings dramatially dereased the antioxidant apaities of flavonoids, whih was learly demonstrated through 5,4 -dihydroxy-7,3,5 - trimethoxyflavone (5). In addition, our data suggested for the first time that stereo onfiguration ould also dramatially affet the antioxidant apaity of flavonoids. The RAC value of (2S,3S)- dihydrokaempferol 3--b-D-gluoside (1) is about 35% higher than its isomer (2R,3R)-dihydrokaempferol 3--b-D-gluoside (2). Nevertheless, how stereo onfiguration affets the antioxidant apaity of flavonoids requires further investigation Anti-inflammatory ativities of flavonoids Table 3 RAC values of ompounds 1 5. Compounds RAC (lmol TE/g) a (2S,3S)-dihyrokaempferol 3--b-D-gluoside (1) ± 52.6 (2R,3R)-dihydrokaempferol 3--b-D-gluoside (2) ± Isovitexin (3) ± Velutin (4) ± ,4 -dihydroxy-7,3,5 -trimethoxyflavone (5) ± 8.8 a Data was expressed as mean ± SD, n =3. NF-jB is one of the prinipal induible transription fators in mammals and has been shown to play a pivotal role in the mammalian innate immune response and hroni inflammatory onditions (Bremner & Heinrih, 2; Li & Stark, 2). Ative NF-jB partiipates in the ontrol of transription of over 15 target genes, inluding the expression of various inflammatory ytokines, hemokines, immunoreeptors, and ell adhesion moleules (Pahl, 1999). NF-jB has thus often been termed a entral mediator of the human immune response (Pahl, 1999). A number of plant-derived substanes inluding ertain flavonoids, suh as querertin (Nair et al., 6) and luteolin (Kim & Jobin, 5) are urrently known as NF-jB modulators. In this study, the ativity and poteny of five flavonoids as NF-jB inhibitors were evaluated by the SEAP reporter assay in RAW-Blue ells. RAW-Blue ells are derived from RAW marophages. They stably express a SEAP gene induible by NF-jB. However, basal expression of the SEAP gene is too low, so it was usually indued by stimuli, suh as lipopolysaharides (LPS). The SEAP protein sereted to the ulture media was measured by a SEAP assay kit as a read-out. Among all five ompounds being sreened, velutin (4) was found to strongly inhibit SEAP seretion in RAW-Blue ells indued by LPS (Fig. 2). Velutin (4) is a flavone that is struturally similar to luteolin, whih was also isolated in aai pulp by our lab (Kang et al., 1). Luteolin is one of the most ommon flavones has its good anti-inflammatory ativities, inluding inhibition of NF-jB ativation (Lopez- Lazaro, 9). In this study, luteolin was adopted as a positive ontrol. Dose response studies of both velutin (4) and luteolin in inhibiting SEAP seretion were further onduted to ompare the effetiveness of these two ompounds (Fig. 3). The IC 5 were alulated as 2. lm for velutin (4) and 12.4 lm for luteolin, respetively. Velutin (4) exhibited muh greater ativity in inhibiting NF-jB ativation than luteolin did. The hemial strutures of velutin (4) and luteolin are very similar with the only differene on two hydroxyl/methoxy groups. Velutin (4) bears two methoxyl groups at 7- and 3 -positions, whereas luteolin has two hydroxyl groups at 7- and 3 -positions. Substitution of methoxyl groups appears to be a signifiant fator that determines inhibition effets. However, exatly how and to what extent the methoxyl groups modulate the inhibition effets of flavones ompounds are yet to be determined. Aai juie has showed effets in the prevention of atheroslerosis in an in vivo animal study (Xie et al., in press). NF-jB ativation is thought to be involved in the initiation and progression of atheroslerosis through its role as a diret regulator of pro-inflammatory and anti-inflammatory genes and as a regulator of ell survival 11 Media LPS µm Fig. 2. Results of the SEAP reporter assay indued by LPS on ompounds 1 5 isolated from aai pulp (initial sreening). The bars represent the mean ± SD (n = 3) from three independent experiments, P <.5. *

6 156 J. Kang et al. / Food Chemistry 128 (11) a velutin (4) d d b Media LPS 5 µm 2.5 µm 1.25 µm.625 µm a luteolin a a b and proliferation (de Winther, Kanters, Kraal, & Hofker, 5; Xanthoulea, Curfs, Hofker, & de Winther, 5). It is well reognised that oxldl plays a ruial role in the initiation and progression of atheroslerosis (Kita et al., 1). Therefore, the inhibitory effet of velutin (4) in oxldl indued NF-jB ativation was also examined. Velutin (4) was found to inhibit NF-jB ativation indued by oxldl (Fig. 4) at onentration as low as 2.5 lm. Sine the oxidation of LDL is a key step in the early stage of atheroslerosis development, this suggests possible athero-protetive effets of this ompound. However, linial validation of these findings is needed. This result may also ontribute to our understanding of why adding aai to high fat diet ould inrease the lifespan of Drosophila melanogaster hallenged by oxidative stress (Sun et al., 1). 4. Conlusions In this study, five flavonoids were isolated and struturally identified from freeze-dried aai pulp. Four of them, (2S,3S)- dihyrokaempferol 3--b-D-gluoside (1) and its isomer (2R,3R)- dihydrokaempferol 3--b-D-gluoside (2), velutin (4) and 5,4 -dihydroxy-7,3,5 -trimethoxyflavone (5), have never been reported from this plant before. The antioxidant apaities of these ompounds were evaluated by RAC assay. The RAC values of the five ompounds varied signifiantly based on their hemial strutures. The methoxyl groups and stereo onfiguration appeared to be important strutural fators that determined their antioxidant apaities. The potential effets of these five flavonoids in inhibiting NF-jB ativation were sreened by SEAP reporter assay. Velutin (4), an unommon flavone, was found to strongly inhibit SEAP seretion in RAW-blue ells indued by LPS or oxldl at low miromole levels, impliating potential anti-inflammatory effets. Further studies are warranted to reveal the mehanisms underlying its inhibitory effets. Media LPS µm 1 µm 5 µm 2.5 µm Fig. 3. Results of dose response experiments of velutin (4) and luteolin from SEAP reporter assay indued by LPS. The bars represent the mean ± SD (n = 3) from three independent experiments. Means with different letters are different (P <.5). a Media oxldl 1 µm 5 µm 2.5 µm Fig. 4. Results of the SEAP reporter assay indued by oxldl on velutin (4). The bars represent the mean ± SD (n = 3) from three independent experiments. Means with different letters are different (P <.5). b Aknowledgments The authors wish to thank Yue Li, Huimin Wang, Danyu Hua and Liangyan Yang from Shanghai Institute of Pharmaeutial Industry, Minjie Xue and Jianjun Wu from Shanghai Institute of Measurement and Testing Tehnology, for MS and NMR measurements; Dr. Steven Talott from Texas A&M University for his ritial omments. The authors appreiated the finanial support of MonaVie LLC (South Jordan, UT) that was required to arry out this study. Referenes Berger, J., Hauber, J., Hauber, R., Geiger, R., & Cullen, B. R. (1988). Sereted plaental alkaline phosphatase: A powerful new quantitative indiator of gene expression in eukaryoti ells. Gene, 66, 1 1. Brasier, A. R. (6). The NF-kappaB regulatory network. Cardiovasular Toxiology, 6, Bremner, P., & Heinrih, M. (2). Natural produts as targeted modulators of the nulear fator-kappab pathway. Journal of Pharmay and Pharmaology, 54, Cao, G., Sofi, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior of flavonoids: struture-ativity relationships. Free Radial Biology and Mediine, 22, Chin, Y. W., Chai, H. B., Keller, W. J., & Kinghorn, A. D. (8). Lignans and other onstituents of the fruits of Euterpe oleraea (Aai) with antioxidant and ytoprotetive ativities. Journal of Agriultural and Food Chemistry, 56, Çitoğlu, G. S., Sever, B., Antus, S., Baitz-Gás, E., & Altanlar, N. (3). Antifungal Flavonoids from Ballota glandulosissima. Pharmaeutial Biology, 41, de Winther, M. P., Kanters, E., Kraal, G., & Hofker, M. H. (5). Nulear fator kappab signaling in atherogenesis. Arterioslerosis, Thrombosis, and Vasular Biology, 25, Del Pozo-Insfran, D., Perival, S. S., & Talott, S. T. (6). Aai (Euterpe oleraea Mart.) polyphenolis in their glyoside and aglyone forms indue apoptosis of HL- leukemia ells. Journal of Agriultural and Food Chemistry, 54, Gallori, S., Bilia, A., Bergonzi, M., Barbosa, W., & Vinieri, F. (4). Polyphenoli onstituents of fruit pulp of Euterpe oleraea Mart (Aai palm). Chromatographia, 59, Hoffmann, A., Natoli, G., & Ghosh, G. (6). Transriptional regulation via the NFkappaB signaling module. nogene, 25, Jensen, G. S., Wu, X., Patterson, K. M., Barnes, J., Carter, S. G., Sherwitz, L., et al. (8). In vitro and in vivo antioxidant and anti-inflammatory apaities of an antioxidant-rih fruit and berry juie blend results of a pilot and randomized,

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