dietary bioactive components

CURRENT DEVELOPMENTS IN NUTRITIONFROM THE AMER ICANSOCIETY FOR NUTR IT ION

© 2018 American Society for Nutrition. All rightsreserved.

Manuscript received September 20, 2017. Initialreview completed March 23, 2018. Revision acceptedMay 27, 2018. Published online July 4, 2018.

Dietary Bioactive Components

Evaluation of a Novel Sequential Release Carotenoid Beadlet Blend in a Simulated GutModel(E08-01)

Kevin W. Gellenbeck,1 Dawna Salter-Venzon,2 and Chun Hu2

1Amway/Nutrilite; and 2Amway R&D, Nutrilite Health Institute

Objectives: When multiple carotenoids are provided at the same time in a nutritionalsupplement form, there can be competition for uptake in the digestive system. Separating theirrelease over time can reduce this interaction and enhance bioavailability. Our objective was toutilize an in vitro gut simulation model to evaluate the release dynamics of a combination ofsequentially released multicarotenoid beadlets. Four beadlets have been designed, composed ofnaturally derived α-carotene (AC), β-carotene (BC), lutein (LU), zeaxanthin (ZX), lycopene (LY),and astaxanthin (AX) that sequentially release their contents over gut transit time to boost overallbioavailability.

Methods: Carotenoids from natural sources were prepared and blended into 4 types ofbeadlets. Two beadlet formulations, AX and a LU+ZX, combination release quickly in thestomach and intestine. Two other beadlet formulations, lycopene and an AC+BC combination,provide controlled release over a period of 4 and 6 h, respectively. Dissolution rates ofthe controlled-release formulations were measured by the manufacturer (Omniactive HealthTechnologies). The release characteristics of a blend of all 4 beadlets were determined in the TIMgastrointestinal simulation model (Triskelion).

Results: Dissolution testing showed that the original prototypes released carotenoids tosolution over a 6-h period in the order AX, LU, ZX, LY, AC, BC. The controlled-release LYproceeded from up to 40% release in the first hour to>75% release in 4 hs. The AC+BC achievedrelease of up to 20% in 2 h, and >80% release in 6 h. Evaluation in the gut simulation modelconfirmedmaximum release and added detail of concentrations over time in the various intestinalcompartments (duodenum, jejunum, ileum).

Conclusions: The results show separation of maximum concentration of the differentcarotenoids through the compartments of the gut simulation model. Combined with results ofa clinical evaluation of a previous version of the beadlet blend, improved bioavailability from asequentially spaced carotenoid mix is suggested.

Funding SourcesFunding provided by Access Business Group International, LLC. Omniactive Health

Technologies provided the beadlet production and in vitro dissolution testing.

Serum and Urinary Metabolites of Allyl Isothiocyanate Derived from Brassica Vegetablesor Authentic Food-Grade AITC (E08-02)

Craig Charron,1 Janet Novotny,1 Harold Seifried,2 and Sharon Ross2

1US Department of Agriculture; and 2National Cancer Institute

Objectives: Glucosinolates are sulfur compounds found mainly in plants of the familyBrassicaceae, such as broccoli, cabbage, and mustard. Glucosinolates are hydrolyzed toisothiocyanates and other breakdown products. Isothiocyanates may reduce cancer risk, and aremetabolized via the mammalian mercapturic acid pathway to conjugated forms that may alsoinhibit cancer. Our objective was to determine whether the source of allyl isothiocyanate (AITC)affects the concentrations of AITC metabolites in human.

Methods: We used an ultrahigh-pressure liquid chromatography triple-quadrupole tandemmass spectrometry method to measure serum and urinary mercapturic acid pathway products of

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AITC. A randomized crossover trial was conducted in which 46 humansubjects consumed a meal that included either 115 µmol of food-gradeAITCor the equivalent in a cabbage/mustard (C/M) portion, and serumand urine samples were collected over 6 h.

Results: AITC-glutathione (AITC-GSH), AITC-cysteineglycine(AITC-CG), AITC-cysteine (AITC-C), and AITC-N-acetylcysteine(AITC-NAC) were detected in serum after both treatments. Themaximum concentration of total AITC metabolites in serum occurredat 3 h and was 591 nM (95% CI: 507, 690) for the AITC treatment and377 nM (95% CI: 323, 440) for the C/M treatment. AITC-NAC wasthe main component, comprising 49 ± 2% and 56 ± 2% (significantlydifferent, P < 0.05) for the AITC and C/M treatments, respectively, at 3h. Maximum urinary excretion rates occurred from 0 to 2 h, and AITC-NAC comprised the majority of the total metabolites excreted in urine.AITC-GSH was not detected in urine.

Conclusions: These data suggest differential absorption andmetabolism of AITC depending on food source, and may thereforedifferentially influence cancer risk.

Funding SourcesSupported by the National Cancer Institute and the US Department

of Agriculture.

High-Fat Diet Impacts Pancreatic Protein Expression: Effects of(-)-Epicatechin Supplementation (E08-03)

Gerardo GMackenzie, Natalia Cortez Penso, Fernando D Villar-real, Elena Daveri, Eleonora Cremonini, and Patricia Oteiza

University of California, Davis

Objectives: Obesity is a major human health problem and itssystemic effects extend to the pancreas. For example, obesity promotesinsulin resistance, has a significant negative impact on the courseof acute pancreatitis, and is associated with an increased risk fordeveloping pancreatic cancer. However, the cellular mechanisms thatmediate these responses are incompletely understood. The objectiveof our study was to identify pancreatic proteins that are differentiallyexpressed in normalmice andmice fed a high-fat diet (HFD).Moreover,we evaluated the effects of (-)-epicatechin (EC) supplementation in theregulation of pancreatic protein expression induced by the high-fat diet.

Methods: Male C57BL/6J mice (5 mice/group) were fed dietscontaining either 10% total calories from fat (control), 60% total caloriesfrom fat (lard) (HFD), or a HFD supplemented with EC (20 mg/kgbody weight) (HFE). After 15 wk of dietary intervention, mice werekilled, and pancreata were collected and subjected to a proteomicsanalysis. Briefly, pancreatic proteins were extracted and subjectedto TMT/iTRAQ profiling. Data were analyzed with iPathwayGuide(impact analysis method).

Results: The proteomics analysis detected the expression of ∼2000pancreatic proteins. When compared to control, HFD led to 1166proteins that were differentially expressed. Among multiple biologicalprocesses/pathways that were found to be the significantly altered,proteins that regulate pancreatic secretion, such as carboxyl ester lipase,carboxypeptidase A1 and protease, serine 2, and proteins involved inthe regulation of reactive oxygen species (ROS) including GlutathioneS-Transferase Pi 1, Peroxiredoxin 2 and Voltage Dependent AnionChannel 1, were the most significantly affected by HFD. Interestingly,

HFE mitigated, in part, the expression of pancreatic secretion proteinsand ROS-generating proteins affected by HFD.

Conclusion: Consumption of HFD alters pancreatic protein ex-pression, and EC supplementation partly mitigates against the HFD-induced effect. These data provide a valuable resource of candidateproteins and pathways that may be altered in obesity, and suggest thebenefit of EC supplementation. Additional studies on mouse pancreassections are warranted to validate these proteomics data.

Funding SourcesThis work was supported by funds from theUniversity of California,

Davis to GGM.

Effects of Fortifying a High-Carbohydrate Cereal Bar withPolyphenol-Rich Berries or Berry Extract on Postprandial Appetiteand Appetite-Mediating Hormone Responses (E08–04)

Claire Whitney,1 Marques Wilson,1 James P Karl,1 Ann Barrett,2Nicole Favreau Farhadi,2 Scott Montain,1 and Tracey Smith1

1USARIEM; and 2NSRDEC

Background: In vitro studies suggest that polyphenols found inberries slow or reduce carbohydrate (CHO) absorption, and effectthat could alter postprandial endocrine responses and appetite ifextant in vivo. However, the impact of fortifying high-CHO foodswith polyphenol-rich berries or berry extracts on appetite-mediatinghormone responses and appetite is not well characterized.

Objectives: The aim of this study was to determine whetherfortifying a high-CHO snack bar with polyphenol-rich freeze-driedblack raspberries or cranberry extract dose dependently impactspostprandial appetite-mediating hormone concentrations and appetite.

Methods: In this randomized, crossover, placebo-controlled trial,20 volunteers (18 M/2 F; 24 ± 5 y; body mass index: 27 ± 3 kg/m2)consumed 1 of 5 bars containing: no berry ingredients (control), 10%or 20% total weight freeze-dried black raspberries [0.7 g (LO-R) or 1.3 g(HI-R) polyphenols], and 0.5% or 1% total weight cranberry extract [0.3g (LO-C) or 0.5 g (HI-C) polyphenols] on trials separated by ≥5 d. Allbars were similar in energy and macronutrient content (451–466 kcal,99–102 g CHO, 6–9 g fiber). Plasma glucagon-like peptide (GLP)-1 andacylated ghrelin concentrations, as well as subjective ratings of hungerand fullness, were measured before and for 3 h after bar ingestion. Adlibitum energy intake at a provided meal was then measured. Dose-dependent effects of the raspberry powder and cranberry extract wereexamined separately by general linear models.

Results: The area under the curve for postprandial GLP-1, acylatedghrelin, hunger, and fullness responses did not differ by raspberryor cranberry dose. A trend for a main effect of dose on ad libitumenergy intake was observed within the raspberry, but not the cranberrytreatment (P = 0.055). Post-hoc testing did not reveal dose-dependenteffects between HI-R and LO-R, but indicated that ad libitum energyintake trended towards being lower in LO-R relative to control (meandifference = 114 kcal; 95% CI: −8, 237 kcal; P = 0.07).

Conclusion:Within the ranges studied, fortifying high-CHO foodswith cranberry extract does not appear to impact appetite. In contrast,fortification with freeze-dried black raspberries may reduce appetite,but this effect does not appear to be dose dependent or associated withchanges in postprandial GLP-1 or ghrelin responses.

CURRENT DEVELOPMENTS IN NUTRITION

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Dietary Bioactive Components 3

Alterations of Plasma Metabolome in Women after Drink-ing Cranberry Juice in a Double-Blinded, Placebo-Controlled,Crossover Design (E08–05)

Shaomin Zhao,1 Haiyan Liu,2 Christina Khoo,2 and Liwei Gu1

1University of Florida; and 2Ocean Spray Cranberries

Background:Biomarkers of cranberry product intake in humans arecurrently lacking, but are needed to verify compliance of participantsin cranberry-related clinical trials. Compliance is unknown in themajority of trials but is presumed to be low due to the astringency ofcranberries. Metabolomics research helps to identify intake biomarkersand providesmissing pieces of a puzzle to explain the physiologic effectsof cranberries.

Objectives: The aim of this study was to investigate plasmametabolome changes after 3 and 21 d of cranberry juice consumption ina double-blinded, placebo-controlled, crossover intervention study, andto compare discriminant metabolites with a previous 3-d open-labeledstudy.

Methods:Adouble blinded, placebo-controlled, crossover interven-tion studywas conducted in 17 healthy youngwomen aged 18–29 ywithnormal body mass index. Plasma samples were collected at baseline,and after 3 and 21 d of consuming double-strength cranberry juice ora placebo juice. Global metabolomic analysis of plasma samples wereconducted by ultrahigh-pressure liquid chromatography Q-Orbitraphigh-resolution mass spectrometry.

Results: Separation of the 2 treatment groups was observed afterboth 3 and 21 d of juice consumption on orthogonal partial least squaresdiscriminant analysis models. One subject was removed from dataanalysis as an outlier. In positive-ionization mode, 103 discriminantmetabolites were found in the S-plot after 3 d of consumption, 19of them were observed to be increasing after cranberry or applejuice consumption in a previous open-label study. Ninety discriminantmetabolites were found after 21 d of consumption, 17 of which wereobserved to be increasing after cranberry or apple juice consumption inthe previous data. In negative-ionization mode, the S-plot revealed 112discriminantmetabolites after 3 d of consumption, 18 ofwhich had beenobserved previously. One hundred and three discriminant metaboliteswere found after 21 d consumption, 17 of which had been observedpreviously.

Conclusion:Alteration of plasmametabolomewas observed amongparticipants after comsuming cranberry juice for 3 and 21 d. Multi-variate analysis verified discriminant metabolites found in a previousopen-labeled study and revealed additional discriminant metabolites ascandidates of cranberry intake biomarkers.

Funding SourcesThis research is funded in part by Ocean Spray Cranberries, Inc.

Effects of Plant Sterols and Stanols plus Pantethine on Lipopro-tein Lipids in Men and Women with Above-Desirable Low-DensityLipoprotein Cholesterol (OR06-01)

Kevin C Maki,1 Belinda Jenks,2 Mary Dicklin,1 Louis Ndife,2Marjorie Bell,1 Cathleen Maki,1 and Wendy Hasse3

1Midwest Biomedical Research/Center for Metabolic and Cardio-vascular Health; 2Pharmavite LLC; and 3Great Lakes Clinical Trials

Objectives: This trial assessed the effects of a dietary supplementcontaining plant sterol/stanol esters + pantethine compared with asupplement of plant sterol/stanol esters alone, incorporated into a dietlow in saturated fat and cholesterol, on low-density lipoprotein (LDL)cholesterol and other aspects of the fasting lipoprotein lipid profile inadults with LDL cholesterol above the desirable range.

Methods:After a 5-wk single-blind diet and placebo lead-in, healthysubjects with LDL cholesterol≥105 and≤195 mg/dL, who were not re-ceiving any lipid-altering drug therapy, were assigned in a randomized,crossover design to receive softgel capsules of a supplement containing1.8 g/d plant sterol/stanol esters alone (SSE; Reducol) or a supplementof 1.8 g/d plant sterol/stanol esters + 600 mg/d pantethine (Pantesin)(SSE + P) for two 6-wk periods. Concentrations of serum lipids andcoenzyme (Co)Q10 and aMEDFICTS dietary assessment questionnairewere analyzed at baseline and at the end of each condition.

Results: A total of 46 subjects (23 men and 23 women) wereenrolled; 43 completed (mean age 48.2 y, mean body mass index 28.3kg/m2). Median baseline concentrations of LDL cholesterol, non-high-density lipoprotein (non-HDL) cholesterol, total cholesterol (TC), HDLcholesterol, TC/HDL cholesterol, and triglycerides (TG) were 119, 147,195, 43.0, 4.2, and 104mg/dL, respectively. Comparedwith SSE, SSE+Pproduced significantly (P < 0.05) larger reductions from baseline inLDL cholesterol (−8.0% compared with −2.4%), non-HDL cholesterol(−4.2% compared with −1.3%), TC (−3.6% compared with −0.21%)and TG (−1.3% compared with+4.8%). HDL cholesterol and TC/HDLcholesterol responses did not differ significantly between conditions.CoQ10 concentrations remained within normal limits. The subset witha MEDFICTS score change below the median during each period hadLDL cholesterol changes of−10.4% compared with−4.2%, for SSE+ Pcompared with SSE, respectively.

Conclusions: These results indicate that 1.8 g/d plant sterol/stanolesters and 600 mg/d pantethine together in a dietary supplementexhibited a greater atherogenic lipoprotein cholesterol–lowering effectthan plant sterol/stanol esters alone. After accounting for dietary drift,effects of plant sterol/stanol esters were consistent with those observedin previous studies and the incremental effect on LDL cholesterol ofpantethine was similar in subjects with and without high dietary drift.

Funding SourcesThis study was funded by Pharmavite LLC (Northridge, CA).

Black Elderberry Extract Improves High-Density LipoproteinFunction in Atherosclerosis-Prone Mice (OR06-02)

Courtney L Millar, Gregory Norris, Christina Jiang, ChelseaGarcia, and Christopher Blesso

University of Connecticut

Objective: Low concentrations of high-density lipoprotein (HDL)cholesterol have been correlated with increased risk for cardiovasculardisease (CVD) in epidemiologic studies. More recently, HDL particlefunctionality, rather than HDL cholesterol, has been suggested tobe a better predictor of CVD risk. However, HDL function may beimpaired during chronic inflammation. Ingestion of bioactive dietarycomponents, such as anthocyanins, may increase both HDL cholesteroland HDL functionality. Black elderberry (Sambucus nigra) containshigh amounts of anthocyanins relative to other edible berries. Weinvestigated whether the consumption of a black elderberry extract


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(BEE) influenced HDL function in apoE–/– mice, which displayimpairments in HDL over time.

Methods:ApoE–/– mice (n= 12/group) consumed ad libitum a low-fat diet supplemented with 0%, 0.25%, or 1% (by weight of diet) BEE(∼37.5–150mg anthocyanins/kg body weight) for 24 wk. Paraoxonase-1 (PON1), an HDL enzyme that prevents lipoprotein oxidation, wasmeasured by its arylesterase and lactonase activities. Ex vivo HDLcholesterol efflux capacity was examined via measuring apolipopro-tein B–depleted serum mobilization of radiolabeled cholesterol fromcAMP-stimulated J774 macrophages. Serum liver enzymes and hep-atic gene expression measures were analyzed as indicators of liverfunction.

Results: PON1 arylesterase and lactonase activities were increasedin the 1% BEE treatment group when compared with control by 32%(P < 0.05) and 45% (P < 0.01), respectively. Both 0.25% BEE and1% BEE strongly increased HDL cholesterol efflux capacity by 64%(P < 0.05) and 85% (P < 0.01) compared with control. Although HDLcholesterol was correlated with PON1 activity and cholesterol effluxcapacity, only supplementation with 1% BEE tended to increase serumHDL cholesterol (P= 0.08). Serum alanine transaminase (P< 0.05) andaspartate transaminase (P< 0.01) activities were reduced with 1% BEE.Analysis of hepatic gene expression revealed that 1% BEE decreased themRNA expression of serum amyloid A (Saa1) (P = 0.04) and tumornecrosis factor α (P = 0.04).

Conclusions: The current study suggests that BEE supplementationdose dependently improves HDL function inmice, while also downreg-ulating the genes involved in the inflammatory process in the liver.

Funding SourcesThis research was supported by USDAAFRI 2015-67018-23145 and

USDA Hatch Grant (CONS00934) to CB.

Supporting Images/Graphs

FIGURE OR06-03-1 Black elderberry extract improves high-density lipoprotein measures.

Barley (1,3)-β-d-Glucan Dietary Supplementation Prevents Car-diac Dysfunction in Obese Mice with Psychosocial Stress by Attenu-ating Myocardial Oxidative Stress (OR06-03)

Jacopo Agrimi,1 Nicole Di Lascio,1,2 Carlotta Baroni,3 GizemKeceli,4 Djahida Bedja,4 and Nicole Di Lascio2

1Unit of Translational Critical Care Medicine, Institute of LifeSciences, Scuola Superiore Sant’Anna, Pisa, Italy; 2Institute of ClinicalPhysiology, National Council of Research, Pisa, Italy; 3Department ofSurgical, Medical, Molecular and Critical Area Pathology, University ofPisa; and 4Division of Cardiology, Johns Hopkins University MedicalInstitutions, MD

Objective: Obesity due to high fat intake and psychosocial stress(PSS) are major determinants of cardiovascular disease (CVD), partic-ularly when combined. Long-term diets enriched with barley (1,3)-β-d-glucan (BBG), an inhibitor of the class I histone deacetylases and anantioxidant, increases post-ischemic survival rate inmice by promotingangiogenesis. However, the impact of a BBG-enriched diet on cardiacdysfunction due to conditions such as high-fat diet (HFD) and PSS,alone or in combination, is unknown. Here, we tested whether sup-plementing HFD-treated mice with BBG prevents HFD- or HFD/PSS-induced cardiac dysfunction by attenuating myocardial oxidativestress.

Methods:Controlmale C57BL/6micewere fedwith 3 different dietsfor 18 wks: 1) standard diet (SD; 10% kcal from fat; n= 9); 2) HFD (58%kcal from fat;n= 9); or 3)HFD+ 3%BBG (HFD+BBG; 58%kcal fromfat; n = 9). All mice were then subjected chronic PSS via a daily (10min) encounter with a male intruder (resident/intruder test, from week16 to week 18). Cardiac function was evaluated by echocardiography


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before and after PSS, whereas left ventricle (LV) tissue was collected atstudy termination to assess reactive oxygen species (ROS) by emissionelectron paramagnetic resonance analysis.

Results: At week 16, body weight was markedly higher in theHFD than in the SD group (+60.1%, P < 0.001); however, BBGsupplementation significantly prevented weight gain (−10% comparedwith HFD, P < 0.05). The addition of PSS significantly worsenedthe LV dysfunction in HFD (LV ejection fraction (LVEF), −25%;LV fractional shortening (LVFS), −33% compared with SD (LVEF,−7%; LVFS, −10%) mice. Conversely, the LV function after PSS wassignificantly improved in HFD + BBG mice (LVEF, +7%; LVFS,+10%) when compared with basal values. HFD and PSS led amarked rise in LV ROS production, as compared with values foundin the hearts of mice receiving SD (+76% P < 0.001). However,this increment was significantly attenuated in HFD + BBG micein which LV ROS emission rose only by 34%. Our study showsthat a regular supplementation of 3% wt/vol BBG with diet preventscardiac functional decay due to the combination of HFD and PSS,likely by countering HFD/PSS-induced myocardial ROS production.These findings may have preventative or therapeutic implicationsfor all CVDs characterized by a cardiac, vascular, or both redoxmilieu.

Funding SourcesUnit of Translational Critical CareMedicine, Istitute of Life Sciences,

Scuola Superiore Sant’Anna, Pisa.

Short-Term Supplementation with Flavanol-Rich Cocoa Im-proves Lipid Profile and Antioxidant Status, and Positively In-fluences Long-Chain n–3 Polyunsaturated Fatty Acids in HealthySubjects (OR06-04)

Sergio Davinelli,1 Graziamaria Corbi,1 Emanuela Ferrara,1Immaculata De Vivo,2 and Giovanni Scapagnini1

1Department of Medicine and Health Sciences “V. Tiberio”, Univer-sity of Molise, Campobasso, Italy; and 2Harvard TH Chan School ofPublic Health, MA

Objectives: We evaluated the short-term effects of a flavanol-richcocoa (FRC) on lipid profile, and selected oxidative stress biomarkerssuch as oxidized low-density lipoprotein (oxLDL), F2-isoprostane, andglutathione (GSH). We also assessed whether FRC increases plasmalevels of n–3 (ω-3) polyunsaturated fatty acids (PUFAs) in healthyindividuals.

Methods: The subjects (n = 48) were randomly assigned to a low-cocoa group (1 g/d; 55 mg flavanols; n = 16), a medium-cocoa group(2 g/d; 110 mg flavanols; n = 16), and a high-cocoa group (4 g/d; 220mg flavanols; n = 16). The samples were collected at baseline, and at1, 2, and 4 h after the first consumption of FRC, and after 4 wk ofsupplementation.

Results: The peak plasma concentration of (–)-epicatechin metabo-lites reached a maximal level (578 ± 61 nM; P < 0.05) at 2 h afteringestion of FRC. After 4 weeks, total cholesterol (−12.37 ± 6.63 nM;P < 0.0001), triglycerides (−3.81 ± 2.45 nM; P < 0.001), plasmaLDL (−14.98 ± 6.77 nM; P < 0.0001), and oxLDL (−95.61 ± 41.69nM; P < 0.0001) decreased in the high-cocoa group compared withbaseline. We also found that plasma high-density lipoprotein (HDL)(+3.37 ± 2.06 nM; P < 0.0001) concentrations increased significantlyin the same group following the intervention. Urinary F2-isoprostanelevels decreased in the medium- (−0.73 ± 0.16 nM; P < 0.0001) andhigh-cocoa (−1.62 ± 0.61 nM; P < 0.0001) groups, whereas only ahigh dose of FRC increased total GSH levels (+209.73 ± 146.8 nM;P < 0.0001). At the end of the 4-wk study, supplementation with ahigh dose of FRC resulted in significant increases in eicosapentaenoicacid and docosahexaenoic acid, as well as a significant reductionin the arachidonic acid:eicosapentaenoic acid ratio (−7.76 ± 4.96;P < 0.0001).

Conclusions: The consumption of FRC may contribute to improv-ing the lipid profile and antioxidant status, as well as influencing themetabolism of PUFAs in healthy subjects. These data extend previousclinical and experimental studies, providing new insights into the healthbenefits of cocoa flavanols.

Funding SourcesThe present study was funded, in part, by a grant from CasaLuker

SA (Colombia).



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FIGURE OR06-04-1 Participantflow diagram + composition of the cocoa + baseline characteristics.

FIGURE OR06-04-2 Plasma levels of (−)-epicatechin metabolites participant flow diagram + composition of the cocoa + baselinecharacteristics.


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FIGURE OR06-04-3 Plasma levels of (−)-epicatechin metabolites.

FIGURE OR06-04-4 Lipid profile.


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FIGURE OR06-04-5 Biomarkers of oxidative stress.


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FIGURE OR06-04-6 AA/EPA ratio.

FIGURE OR06-04-7 Lipid profile.


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FIGURE OR06-04-8 Biomarkers of oxidative stress.

FIGURE OR06-04-9 AA/EPA ratio.

A Muscadine Grape Extract Improves Hypertension-InducedDiastolic Dysfunction and Cardiac Damage through Anti-Fibroticand Anti-Oxidant Mechanisms (OR06-05)

Pooja D Patil, Brian Westwood, Jasmina Varagic, PatriciaGallagher, and Elisabeth Tallant

Wake Forest School of Medicine, NC

Objective: The aim of this study was to determine whether a mus-cadine grape skin and seed extract (MGE) will improve hypertension-induced cardiac damage in a prevention model.

Methods: Sprague-Dawley rats (male, 8 wk old) were not treated(Control), administered MGE at 0.2 mg total phenolics/mL drinkingwater (MGE), administered 24 μg · kg–1 · h–1 Ang II via an osmoticminipump (Ang II) to induce hypertension, or treated with Ang II andMGE (Ang II/MGE). Rats receiving MGE were pretreated for 1 wkprior to Ang II treatment. Blood pressure was measured by tail cuffplethysmography and cardiac function by echocardiography.

Results: MGE had no effect on blood pressure in normotensiveor hypertensive rats. MGE ameliorated Ang II-induced diastolicdysfunction (E/E′ ratio: 19.9 ± 0.8 Control, 28.1 ± 1.1 Ang II,22.3 ± 2.0 Ang II/MGE; n = 8; P < 0.05); MGE alone had noeffect on any parameters. MGE improved the Ang II increase in aorticpulse wave velocity (2.4 ± 0.3 Control, 6.8 ± 1.4 Ang II, 3.6 ± 0.5Ang II/MGE; P < 0.05), suggesting that the extract reduces vascularstiffness. Cotreatment with MGEmarkedly reduced the Ang II increasein cardiomyocyte cross-sectional area (P < 0.01), interstitial fibrosis(P< 0.05), and collagen III deposition (0.9± 0.2% Control, 6.8± 1.0%Ang II, and 2.8 ± 0.4% Ang II/MGE; P < 0.01). TGFb is a cytokinemediating fibrosis through regulating Smad signaling. MGE preventedthe Ang II increase in TGFbmRNA (1.0± 0.1 Control, 2.0± 0.4 Ang II,1.1 ± 0.2 Ang II/MGE) as well as Smad2 activation in cardiomyocytes(26.1± 6.0Control, 53.8± 9.6Ang II, 27.6± 5.5Ang II/MGE;P< 0.05)and fibroblasts (2.3 ± 1.1 Control, 11.7 ± 4.9 Ang II, 2.2 ± 0.5 AngII/MGE; P < 0.05). NADPH oxidase (Nox) is a primary contributorto cardiac oxidative stress and excessive amounts of reactive oxygenspecies activate fibrosis. MGE reduced p22phox mRNA (1.0 ± 0.1Control, 1.9 ± 0.1 Ang II, 1.0 ± 0.1 Ang II/MGE; P < 0.0001), anessential subunit of Nox, and Nox4 (0.24 ± 0.01 Control, 0.33 ± 0.01Ang II, 0.27 ± 0.01 Ang II/MGE; P < 0.0001). Finally, Ang II increased


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circulating 8-hydroxy-deoxyguanosine, ameasure of oxidative stress, by101%, which was reduced by MGE to 26%.

Conclusions: These results suggest that MGE may serve as amedical food to protect the heart from hypertension-induced diastolicdysfunction, hypertrophy, fibrosis, and oxidative damage.

Funding SourcesChronic Disease Research Fund.

Milk Polar Lipids Reduce Cardiometabolic Risk Factors inPostmenopausal Women: A Randomized Double-Blind ControlledTrial (OR06–06)

Marie-Caroline Michalski,1 Cécile Vors,1 Laurie Joumard-Cubizolles,2 Emmanuel Combe,1 Jocelyne Drai,1 Ketsia Raynal,3Patrice Gaborit,3 Florent Joffre,4 Geneviève Gesan-Guiziou,1Lemlih Ouchchane,5 Martine Laville,1 Corinne Malpuech-Brugère,2 and Stéphanie Lambert-Porcheron6

1INRA—Institut National de la Recherche Agronomique;2Université Clermont Auvergne, INRA, UNH, Unité de NutritionHumaine, CRNH Auvergne, Clermont-Ferrand, France; 3ACTALIA,Surgères, France; 4ITERG, Pessac, France; 5Université ClermontAuvergne, UMR 6602 CNRS/UCA/SIGMA, IP, Clermont-Ferrand,France; and 6CRNH-RA, CENS, Oullins, France

Objective: Disturbances of both fasting and postprandial lipidprofiles are recognized as cardiovascular risk factors. Nutrition plays amajor role in their modulation, notably in postmenopausal women atrisk of cardiovascular disease (CVD). Interest has recently grown onthe potential benefits of milk polar lipids (MPLs), which are rich insphingomyelin. However, effects of MPL supplementation in humanshave been inconsistent, often due to trials being performed in healthysubjects or by increasing energy intake. We hypothesized that isolipidicenrichment of the diet with MPL via a realistic dairy product couldimprove lipid markers of cardiovascular risk.

Methods:Weperformed a double-blind randomized controlled trialin 58 postmenopausal overweight womenwith high-density lipoprotein(HDL) cholesterol <1.6 mM. They were subjected to the followingregimes: 1) a 4-wk dietary intervention with daily consumption of adairy product containing 12 g of milkfat randomly including either0 g (control, n = 19), 3 g (n = 19), or 5 g (n = 20) of MPLs; and2) 8-h postprandial explorations after a test meal, before and afterintervention (i.e., 2 visits). Lipid markers were measured in plasma andin the chylomicron-rich fraction. The effect of MPL dose was testedthrough a linear mixed model (SAS) seeking differences between visitsand between the 3 intervention groups.

Results: Daily 4-week consumption of milkfat enriched with 5g MPLs decreased significantly the plasma concentrations of bothfasting and postprandial total cholesterol (P < 0.001) and plasmatriglycerides (P < 0.001), decreased fasting low-density lipoprotein(LDL) cholesterol (P < 0.001) while increasing HDL cholesterol(P < 0.05), and decreased apolipoprotein B/A1 ratio (P < 0.001).Effects were significantly different (P < 0.05) from those of the control,which did not modify lipid profile. MPL dose impacted chylomicron-cholesterol and chylomicron-triglycerides (P < 0.01), with decreasedconcentrations observed in the 5 g MPL group (P < 0.01), suggestinglower intestinal absorption and increased remnant clearance.

Conclusions: These data suggest that a dietary strategy based onmilk lipid quality, including MPLs, can contribute to improving thecardiometabolic health of postmenopausal women.

Funding SourcesANR (FrenchNational ResearchAgency), PHRC-I (FrenchHospital

Program for Clinical Research), CNIEL (French Dairy InterbranchOrganization).

Curcumin Supplementation Improves Heat Stress–Induced Car-diac Injury of Mice: Physiologic and Molecular Mechanisms(OR06–07)

Yong Chen,1 Yuechao Du,1 Chao-Qiang Lai,2 and Lirong Shen1

1ZhejiangUniversity, China; and 2USDAARSNutritional GenomicsLaboratory, JM-USDA Human Nutrition Research Center on Aging atTufts University, MA

Objectives: The aim of this study was to examine the effect ofcurcumin (CUR) on cardiac function of mice exposed to heat stress(HS).

Methods: Male C57BL/6J mice 6 wk old were randomly dividedinto 6 groups (n = 8 each): control (CK), heat treatment (HT), aspirin(AP), low-dose CUR (CUR50), medium-dose CUR (CUR100), andhigh-dose CUR (CUR200). The 3 CUR groups were fed daily bygavage, 50, 100, and 200 mg CUR/kg for 4 wk. The AP group wasadministered 1 mg AP/kg orally 2 h before HS. Except for CK, theother groups were exposed once to HS at 41°C for 20 min. Immediatelyafter HS, the blood pressure (BP), rectal temperature (TM), and heartrate (HR) of all mice were recorded. Four hours later, the mice werekilled under deep anesthesia. Cardiac Troponin I (cTnI) in serumand Angiotensin II (Ang II) in cardiomyocytes were measured byELISA assays. mRNA expressions of Angiotensin receptor 1 (At1) incardiomyocytes were analyzed by quantitative reverse transcriptase-polymerase chain reaction.

Results:Compared with CK, the HR of the HT group was decreasedby 30.2%, whereas the HRs of AP, CUR50, CUR100, and CUR200were increased by 25.0%, 49.5%, 39.6%, and 42.7%, respectively. TheBP in the HT group was increased (P = 0.0075), but no significanteffect was found in the other groups. The TM of HT and CUR50were raised to 41.5°C and 40.9°C, whereas mice in AP, CUR100, andCUR200 groups showed less TM increase (39.8°C, 40.0°C, 39.3°C,respectively) than the HT group (41.5°C, P<0.05), indicating that CURcould mitigate the physiologic stress induced by HS. In addition, thecardiac damage biomarker (cTnI) in HT was increased significantly(P<0.001) compared with that of CK. However, pretreatment withCUR normalized cTnI level significantly (P<0.05) compared with theHT group. Similarly, CUR attenuated the increase of Ang II inducedby HS. Moreover, At1, highly increased by HS, was reduced by CUR,elucidating the molecular mechanism of CUR to improve HS-inducedcardiac injury.

Conclusions: These observations provide evidence that CUR couldalleviate physiologic stress and cardiac damage caused by HS.

Funding SourcesThe National Key Research and Development Program of China

(2017YFC1601700), National Natural Science Foundation of China(31271848), Foundation of Fuli Institute of Food Science of Zhejiang


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University (KY201404), Foundation from the USDA, AgricultureResearch Service (58-1950-9-001).

DailyConsumptionof 20–30 gGroundFlaxseed ImpairsVitaminB-6 Status in a Randomized, Controlled, Crossover Trial in Adultswith Above-Optimal Plasma Low-Density Lipoprotein Concentra-tions (OR06-08)

Heather J Blewett,1 Jay Petkau,1 Li Ren,1 Randy Guzman,2Thomas Wolever,3 and Michel Aliani2

1Agriculture and Agri-Food Canada; 2University of Manitoba,Canada; 3University of Toronto, Canada

Objectives: Flaxseed contains the vitamin B-6 antagonist linatine.We aimed to determine the effect of flaxseed (0, 20, and 30 g) on thefollowing markers of B-6 status: plasma homocysteine (HCY; whichwould increase with impaired B-6 status), and aspartate aminotrans-ferase (AST; a B-6–dependent enzyme the activity of which activitywould decrease with impaired vitamin B-6 status).

Methods: A multisite, randomized, double-blind, controlled,crossover study was conducted at the IH Asper Clinical ResearchInstitute in Winnipeg (n = 34) and GI Labs Inc. in Toronto (n = 29)(clinicaltrials.gov NCT01821131). Sixty-three (32 men, 31 women)healthy nonsmokers, age 30–65 y, body mass index 18.5–40.0 kg/m2,low-density lipoprotein (LDL) 2.6–5.0 mmol/L, not taking cholesterolor blood-pressure medication, completed the trial, which had 3phases, each lasting 4 wk, separated by 4–8 wk. During each phase,participants ate 1 muffin/d containing either 0 (control), 20, or 30 gground flaxseed. The order of treatments was random. Plasma wascollected at the start and end of each phase. Plasma HCY and ASTwere measured on a cobas c111 clinical chemistry analyzer. SAS (9.4)was used for analysis of covariance, with the use of Proc GLM withthe following variables in the model: treatment, order of treatment,participant, baseline value, interaction between treatment, and order oftreatment.

Results: There was a significant (P = 0.04) effect of flax on finalHCY concentrations, with 30 g flax increasing HCY by 66% comparedwith 0 g flax (17.3± 1.9 compared with 10.4± 1.9 µmol/L). Final HCYconcentrations with 20 g flax (12.7 ± 1.9 µmol/L) were not differentfrom the other treatments. The 30-g flaxseed treatment resulted in amean plasmaHCY in the hyper-homocysteinemic range (>15µmol/L).AST decreased significantly (P< 0.001) after 30 and 20 g flax by 9–13%compared with 0 g flax (20.0 ± 0.4 and 21.0 ± 0.4 U/L compared with23.0 ± 0.4 U/L, respectively).

Conclusions: These are the first data in humans to show thatconsumption of ground whole flaxseed at doses below the HealthCanada–approved LDL cholesterol–lowering health claim negativelyaffects B-6 status, causing an increase in a risk factor for cardiovasculardisease.

Funding SourcesAgriculture and Agri-Food Canada.

Green Tea Reduces Metabolic Endotoxemia in NonalcoholicSteatohepatitis in Association with Altered Relative Abundance ofBile Acids and Phosphatidylcholine Metabolites (OR22-01)

Geoffrey Y Sasaki, Jinhui Li, Morgan J Cichon, Ken M Riedl,Rachel E Kopec, and Richard Bruno

The Ohio State University

Objectives: Green tea extract (GTE) lowered hepatic nucleartranscription factor κB (NF-κB)–mediated inflammation during nonal-coholic steatohepatitis (NASH) in association with increased intestinaltight junction proteins that limit metabolic endotoxemia. From thesemice, our objective was to identify shifts in the global hepaticmetabolome that are potentially responsible for alleviating metabolicendotoxemia and NF-κB–mediated inflammation during NASH.

Methods: Male C57BL/6J mice were fed a low-fat (LF) or high-fat (HF) diet for 12 wk to induce NASH. They then continued onthese diets supplemented with 0 or 2% GTE (n = 10/group) for anadditional 8 wk prior to assessing hepatic metabolomics profiles byliquid chromatography-quadrupole time-of-flight mass spectrometry.

Results: Principal component analysis indicated that GTE in HF-fed mice restored the hepatic metabolome that was otherwise shiftedaway from LF-fed mice. Compared with HF controls, 129 metaboliteswere altered (≥2-fold; P < 0.01) in response to GTE. Featuresthat were putatively identified include phosphatidylcholine catabolites(e.g., lysophosphatidylcholine, glycerophosphocholine) and primaryand secondary bile acids (e.g., muricholic acid, sulfoglycolithocholate).GTE in HF mice decreased (P < 0.05) the relative abundance ofphosphatidylcholine catabolites that were otherwise increased in HFcontrols.

Compared with HF controls, GTE increased bile acids and de-creased hepatic cholesterol to levels not different from LF mice.Phosphatidylcholinemetabolites were positively correlatedwith hepaticphosphorylated p65 and malondialdehyde (r = 0.58–0.63). Bile acidswere inversely correlated with serum endotoxin (r = −0.68 to −0.71)and phosphorylated p65 (r = −0.47 to −0.56). Bile acids were alsopositively correlated with mRNA expression of duodenal occludin,zonula occluden-1, and claudin-1 and ileal occludin and claudin-1(r = 0.36–0.46).

Conclusions: These findings suggest that metabolic shifts by GTEalleviate NF-κB inflammation in NASH by enhancing gut barrierfunction in association with improvements in bile acid metabolism andlimiting lysophosphatidylcholine-mediated hepatic injury.

Funding SourcesFoods for Health, a focus area of the Discovery Themes Initiative at

The Ohio State University, and the Food Innovation Center.

Bovine Milk Exosomes and their miR-30d Cargo Cross thePlacenta and Contribute Toward Embryo Development in C57BL/6Mice (OR22-02)

Mahrou Sadri, Jiang Shu, Juan Cui, and Janos Zempleni

University of Nebraska-Lincoln

Background: Exosomes are nanoparticles that play an essentialrole in cell-to-cell communication through the delivery of regulatorymolecules such as microRNAs from donor cells to recipient cells. Wedemonstrated that dietary exosomes andmicroRNAs in bovinemilk arebioavailable and their depletion elicits phenotypes in nonbovine species.

Objectives: This study had the following aims: 1) to assess theaccumulation of bovine milk exosomes (BMEs) in murine placenta andembryos; 2) to assess gene expression patterns in the placenta of mice


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fed milk exosome- and RNA-depleted (ERD) and exosome- and RNA-sufficient (ERS) diets; and 3) to assess the effects of ERD and ERS onembryo development.

Methods: The placental and embryonic accumulation of BMEswas assessed by administering fluorophore (DiR)-labeled BME topregnantmice (day 16.5) by oral gavage; similar studies were conductedby transfecting BMEs with a synthetic, fluorophore (IRDye)-labeledmicroRNA implicated in adhesion during embryo implantation, miR-30d, and administering the BMEs to pregnant mice on day 16.5 byoral gavage. Controls received unlabeled BME. Tissue fluorescence wasmeasured with a LI-COR imager. Gene expression was assessed inplacentas from mice fed AIN-93G–based ERD and ERS diets on day14.5 of pregnancy by RNA sequencing and KEGG pathway analysis.Embryo development and survival were assessed based on litter size asa marker. ANOVA, Dunnett’s post-hoc test, and paired and unpaired ttests were used for statistical analysis, with P

FIGURE OR22-02-1 Administration of DiR-labeled BME to mice; gestational day 16.5. Panels depict placentas [A, unlabeled BME(control); B–D, DiR-labeled BME, n = 3 controls, 3 treatments; E, miR-loaded BME] and embryos [F, unlabeled BME (control); G–I,DiR-labeled exosomes, n = 3 controls, 3 treatments); J, miR-loaded BME].

FIGURE OR22-02-2 Densitometry analysis of BME in murineembryos and placenta (DiR-labeled BME minus unlabeled BME);∗P < 0.05, n = 3/group).

Results: Both DiR-labeled and miR-loaded BME accumulatedin placentas and embryos (Figure 1); accumulation was moderatelyhigher in placenta than in embryo (Figure 2). Seventeen genes weredifferentially expressed in the placentas from ERD and ERS mice(>2-fold change, P e.g., Col1a1/2 and Tagln(Figure 3). The litter sizeproduced by ERS/ERS breeders was twice the size of the litters in othergroups(Figure 4).

Conclusion: BME and their microRNA cargo accumulate inmurineplacenta and embryos and elicit a gene expression profile conduciveto embryo survival and implantation. Dietary depletion of exosomescauses a decrease in litter size. Future studies will assess implantationsites in uterus/placenta and establish a cause-and-effect relationshipthrough microRNA knockout.

Funding SourcesNIFA 2015-67017-23181, NIFA 2016-67001-25301, NIH

1P20GM104320, Gerber Foundation, Egg Nutrition Center, and USDAHatch Act and W3002.


FIGURE OR22-02-3 mRNA expression analysis in placenta fromC57BL-6 mice fed ERS or ERD diet (n = 3/group).


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FIGURE OR22-02-4 Size of litters born to C57BL/6 breeders fedERS and ERD diets; ∗P < 0.05 compared with control, n = 8.

A Randomized Crossover Intervention Study of the Effect ofCow’s Milk Consumption on Endogenous Sex Steroid HormoneLevels in Postmenopausal Women (OR22–03)

Karin B Michels,1 Nadine Binder,1 Frédérique Courant,2 AdrianA Franke,3 and Anja Kaiser-Osterhues1

1University of Freiburg, Germany; 2Laboratoire d’Etude des Résiduset Contaminants dans les Aliments, France; and 3University of HawaiiCancer Center

FIGURE OR22-03-1 Study design.

Objectives: Current cow’s milk production practices introduceconsiderable levels of pregnancy hormones into the milk. Weconducted a randomized crossover intervention study to evaluatewhether consumption of cow’s milk affects urinary excretion ofsex steroid hormones and their metabolites and whether any effectmay depend on the fat content of the milk (1.5% compared with3.5%).

Methods: A total of 110 postmenopausal women were asked toconsume 1 L of semiskimmed milk (1.5% fat) per day for 1 wk and 1L of whole milk (3.5% fat) per day for 1 wk, intersected by wash-outperiods (Figure 1). Sex steroid hormone levels were measured in 24-hurine samples collected at the end of each intervention and wash-outperiod.

Results: Estrogens, androgens, and progesterone were detectedin the examined milk samples used for our intervention, withprogesterone levels being particulalry high. Although a very highproportion of the estrogens were conjugated, only small proportionsof the androgens and progesterone were conjugated. Of the 110women included, 109 (99%) completed all aspects of the study. Milkconsumption resulted in a significant increase in urinary estrone(E1) excretion, whereas estradiol (E2), estriol (E3), and 16ketoE2excretion only increased after semiskimmed milk consumption.Urinary pregnanediol glucuronide excretion was not significantlyaffected.

Conclusions: Consumption of cow’s milk increases urinary ex-cretion of E1 in humans. Ingestion of semiskimmed milk appearsto also raise E2, E3, and 16ketoE2 excretion, but future studiesneed to confirm these associations. Collecting repeated 24-h urinesamples to assess biomarkers was associated with excellent studyparticipation.

Funding SourcesSupported by research grant MI 1461/3-1 from the German

Research Foundation.



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Genistein Stimulates AMPK Phosphorylation via GPR30 and/orPDE3A Stimulating theMitochondrial Capacity in C2C12Myotubes(OR22–04)

Sarai Vasquez-Reyes,1,2 Nimbe Torres,2 Lilia G Noriega,2 andArmando Tovar2

1UNAM, Mexico; and 2Instituto Nacional de Ciencias Medicas yNutricion SZ, Mexico

Objectives: Genistein is known to stimulate health effects, par-ticularly in skeletal muscle, by promoting AMPK phosphorylation.However, the mechanism of its activation has not been clearlyestablished. Thus, the aim of the present study was to assess themechanism by which genistein promotes AMPK phosphorylation andits effect on mitochondrial capacity.

Methods: We studied the effect of genistein on AMPK phosphory-lation through the use of C2C12 myotubes. We then evaluated whetherAMPK phosphorylation was stimulated by forskolin or 8-bromo cAMP.Furthermore, we assessed whether genistein was able to activate thesignal transduction pathway activated by cAMP to promote AMPKphosphorylation via inhibition of phosphodiesterase PDE3A, or byactivating the G-coupled receptor GPR30. Therefore, we measuredwhether genistein modified PDE3A or GPR30 gene expressionassessed by quantitative polymerase chain reaction and western blotin C2C12 myotubes. Finally, we measured the oxygen consumptionrate (OCR) and the acidification rate (ECAR) of the medium withthe Seahorse system, incubating differentiated C2C12 cells withdifferent concentrations of genistein (0.1, 0.3 1, 3, 10, 30 µM) for1 h.

Results:Genistein (0.1–30 µM) stimulated AMPK phosphorylationafter 1 h of incubation.We have evidence that genistein increases cAMPlevels, and in our study either forskolin or 8-bromo cAMP were ableto stimulate AMPK phosphorylation. We demonstrated that genisteinupregulated the expression of GPR30 but not PDEA3, suggesting thatthe potential increase of cAMP can be mediated via GPR30. Finally weobserved that the increase in AMPK phosphorylation was associatedwith an increase in both OCR and ECAR after 1 h of stimulationwith genistein, indicative of an increase in glycolysis andmitochondrialcapacity.

Conclusions: Our evidence suggests that genistein can increasethe oxidative capacity of C2C12 myotubes by increasing AMPKphosphorylation, possibly via GPR30, leading to an increased gly-colytic and mitochondrial capacity. This may explain how genisteincan improve insulin sensitivity, particularly by increasing fatty acidoxidation.

Funding SourcesInstituto Nacional de Ciencias Medicas y Nutricion SZ, CONACYT.

Hydroxytyrosol: A Promising Bioactive against Parkinson Dis-ease (OR22-05)

Mª Carmen Garcia-Parrilla, Ruth Hornedo-Ortega, Ana BCerezo, and Ana M Troncoso

Universidad de Sevilla, Spain

Objectives: Parkinson disease (PD) is the most common neu-rodegenerative movement disorder, and is currently incurable. Theprincipal pathologic hallmark is the presence of extracellular depositsof abnormal aggregated α-synuclein (αsyn) in the brain (Lewy bodies),which trigger toxicity and neuronal death. Hydroxytyrosol (HT), aphenolic compound present in virgin olive oil and olives, and also ingrapes and wine, is widely known for its healthy properties includingits neuroprotective capacity. If dietary HT is able to counteract αsyntoxicity, it could serve as a potential neuroprotector against PD. Thus,the aim of this work was to evaluate the potential inhibitory anddestabilizing effect of HT on αsyn aggregation.

Methods: We measured the kinetics of αsyn fibril formationand αsyn destabilization with the use of the Thioflavin T (ThT)assay. The experiments were performed with ThT protein, whichemits fluorescence when assembled with misfolded proteins, and isconsidered a model marker to monitor the kinetics of fibril formation.αsyn and αsyn preformed fibrils (70 µM) were incubated in thepresence of HT (25, 50, 100, and 200 μM). Fluorescence emission datawere recorded every 2 h for 6 d under continuous agitation (αsyn) andwithout agitation (fibrils). Samples from the ThT assay (αsyn with HTat 25, 50, 100, 200 μM) were placed on carbon-coated formvar grids,incubated, and then viewed in a transmission electron microscope at80 kV.

Results: All tested HT concentrations (25–200 µM) were active atinhibitingαsyn fibril formation(Figure 1). This compound inhibited thefibrillation process between 70% and 89%. These results were confirmedwith images obtained by transmission electron microscopy(Figure 2A–E). When αsyn was incubated alone, the fibrils were longer andcompact. Conversely, thin and short fragments were observed followingcoincubation with all HT concentrations tested. Furthermore, HTalso is capable of destabilizing the preformed fibrils of αsyn. Wehave obtained destabilization percentages of ∼30–80% (Figure 3).Transmission electron microscopy images (Figure 4A–E) confirmedthis effect.

Conclusion: HT showed significant in vitro effect in the mech-anisms involved in the progression of PD, and can be considered apromising bioactive compound against neurologic disorders.

Funding SourcesMinisterio de Economía y Competitividad (project code AGL2016-

77505-C3-2-R).



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Effects of Soy-Based Diets on Neurologic and Metabolic Pheno-types (OR22-06)

Cara J Westmark, Mikolaj Filon, Pamela R Westmark, PatriciaMaina,DavidWNelson, BrianCRay, Lauren I Steinberg, TaralynMWilmer, Chi-Liang Eric Yen, and Chrysanthy Ikonomidou

University of Wisconsin-Madison

Objective:During the course of studying seizures in Fmr1KOmice,a rodent model for the developmental disability fragile X syndrome,we serendipitously discovered that the type of rodent feed (casein-based purified ingredient diet compared with grain/soy-based chow)significantly affects seizure propensity. Our objectives herein wereto determine if diet affects growth metrics or additional neurologicphenotypes and biomarkers.

Methods: The study design included the following elements: 1)retrospective analysis of prior seizure datasets containing body weightmetrics to ascertain differences in weight as a function of age,diet (casein-based purified ingredient compared with grain/soy-based

chow), and genotype (WT and Fmr1KO); 2) comparison of infantgrowth metrics in response to casein- and soy-based infant formulabased on the use of the CDC Infant Feeding Practices Study II (IFPSII)dataset; and 3) prospective evaluation of weight gain and plasmabiomarker expression in WT and Fmr1KO mice in response to casein-and soy-based infant formula diets.

Results: Juvenile mice fed chow throughout gestation and postnataldevelopment exhibit increased weight gain compared with mice fed acasein-based purified ingredient diet. The effects are more pronouncedin Fmr1KO mice. Adolescent WT and Fmr1KO mice weaned onto soy-based infant formula diet exhibit increased weight gain compared withmice weaned onto a casein-based infant formula diet. Adult Fmr1KO

mice transferred to soy infant formula diet also exhibit excessiveweight gain. Thus, at the systems level, our findings indicate that theconsumption of grain/soy-based diets increases weight gain in miceirrespective of age, with themost pronounced effects in juvenile Fmr1KO

mice. Retrospective analysis of the CDC IFPSII dataset indicates thatconsumption of soy-based infant formula may be associated with


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increased body mass index in female infants exclusively fed formulathrough 12 mo of age. At the molecular level, the expression of severalmouse plasma proteins, including the adipose hormone leptin and theamyloid-β–degrading enzyme neprilysin, are altered in response todiet.

Conclusions: Consumption of single-source grain/soy-based dietsmay exacerbate adverse neurologic phenotypes and accelerate growthin mice, particularly in fragile X.

Funding SourcesUniversity of Wisconsin ICTRFRAXA Research Foundation,

NICHD, RayBiotech

Role of Gut Microbiota–Derived Polyphenolic Metabolites inNeurodegenerative Disorders Involving Protein Misfolding andC9orf72 Expansion (OR22–07)

Giulio M Pasinetti,1 and Lap Ho2

1Mount Sinai School of Medicine, NY; and 2Icahn School ofMedicine at Mount Sinai, NY

Objective: There is growing evidence that in many neurodegen-erative disorders, cell-to-cell transmission of a pathologic misfoldedprotein, such as misfolding of αsynuclein (αsyn) in Parkinson disease(PD), may be a vehicle for the spreading of pathology throughoutthe brain. This misfolded protein, or seed, further induces misfoldingof native proteins within the cell. Pathologic misfolded proteins mayexist in diverse conformations with distinct cellular and biochemicalproperties.We investigate whethermicrobiota-derivedmetabolitesmayhelp to attenuate the misfolding of αsyn and thereby promote resilienceagainst PD phenotypes.

Methods and Results: We identified 6 biologically available gutmicrobiota–derived compounds (GMP10, GMP11, GMP26, GMP28,GMP39, and GMP44) for investigation. With the use of independentin vitro protein aggregation assays (e.g., photoinduced crosslinking ofunmodified proteins assay, thioflavinT, fluorescence assay, and electronmicroscopy) we demonstrated that 3 of the compounds (GMP26,GMP44, GMP28) potently inhibit aggregations of monomeric αsynm(or monomeric βamyloid peptides) into neurotoxic protein aggregates,in vitro. Based on evidence linking the c9orf72 gene with expansions ofGGGGCC hexanucleotide repeats and PD, as well as amyotrophic lat-eral sclerosis (ALS) and frontotemporal dementia (FTD), we continueto test the neuroprotective ability of our compounds in vivo with the useof a Drosophila model with overexpression of GGGGC hexanucleotiderepeats. Overexpression of 30 GGGGCC repeats in the Drosophila eyecauses age-dependent photoreceptor neurodegeneration. We treatedDrosophila by mixing individual test compounds into the food andfound all 6 compounds significantly suppressed eye degeneration at10 μM, with compounds GMP26 and GMP11 almost completelysuppressing the eye phenotype. The comparative efficacy of the6 compounds are GMP26 = GMP11 > GMP39 > GMP10 >

GMP44 > GMP28.Conclusions: Outcomes from our studies link gut microbiota with

mechanisms underlying PD and suggest the feasibility of developingGMP26 as a means to simultaneously target both αsyn misfolding andC9orf72 expansion to increase the likelihood of therapeutic efficacy inPD, ALS, and FTD patients with C9orf72 expansion.

Funding SourcesFunding was provided by the P50 AT008661-01 from the National

Center for Complementary and Integrative Health (NCCIH) andthe Office of Dietary Supplements (ODS), NIH R01 MH090264,NIH R01 MH104559, NSF81200862, and support from the AltschulFoundation.

Effect of Genistein Consumption in Subjects with Obesity andInsulin Resistance on Serum Metabolomics and Gut Microbiota(OR34-01)

Armando RTovar, Einar Godinez-Salas,Maria del Rocio Guizar-Heredia, Liliana Arteaga-Sanchez, Edgar Pichardo-Ontiveros,Gonzalo Torres-Villalobos, Monica Sanchez-Tapia, MarthaGuevara-Cruz,and Nimbe Torres

Instituto Nacional de Ciencias Medicas y Nutricion SZ, Mexico

Objectives: Studies with animal models have shown that genistein,a soybean isoflavone, improves insulin resistance by stimulating AMPKin skeletal muscle. Evidence in humans indicates that genistein canimprove insulin sensitivity. Recent studies in experimental animalssuggest that the effects of genistein can be mediated in part bymodification of the gut microbiota. The aim of the study was to assesswhether genistein consumption for 2 mo improves insulin sensitivityin subjects with obesity and insulin resistance by modifying the gutmicrobiota and metabolomics.

Methods and Results: We conducted a double-blind randomizedclinical trial in 34 subjects with obesity and insulin resistance. Subjectsconsumed a diet reduced by 500 kcal from their habitual diet for 2 wkand then continued with the same diet plus treatment with placeboor genistein. A clinical, anthropometric and biochemical evaluationwas conducted. The gut microbiota was evaluated with the Illuminaplatform, the serum metabolomics was evaluated by HPLC-massspectrometry, and gene expression in a skeletal biopsy by microarrays(Affymetrix). The results showed that genistein consumption signifi-cantly decreased body weight, body mass index, waist circumference,fat mass, triglycerides, C-reactive protein, leptin, and AUC insulin,and increased lean body mass. These changes were accompanied byan increase in the Verruccomirobia and Bacteroidetes phylum, leadingin a decrease in serum lipopolysaccharide (LPS). The metabolomicsprofile showed that genistein increased the concentration of severalmicrobiome-derived phenolicmetabolites, several fatty acids, includingdicarboxylate and monohydroxy fatty acids, as well as acyl-carnitinesand lysolipid metabolites.

Conclusions: The results of this study demonstrated that consump-tion of the bioactive compound genistein improves insulin sensitivity inpart by modifying the gut microbiota, reducing metabolic endotoxemiamediated by LPS, and the presence of specify metabolites.

Funding SourcesCONACYT.

Green Tea Polyphenols Modify Gut-Microbiota–Dependent En-ergy and Micronutrient Metabolism in Rats (OR34-02)

Jun Zhou,1 Lili Tang,1 Leslie Shen,2 and Jia-Sheng Wang1

1University of Georgia; and 2Texas Tech University


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Objects:Microbiome analysis demonstrated that green tea polyphe-nols (GTPs) could modify gut-microbiota community structure andenzyme orthologs in rats. How these modifications affect host healthis largely unknown. Metabolomics analysis, therefore, was conductedto explore the metabolic profiles and changes of the gut-microbiota–dependent metabolisms, as well as their potential health outcomes inrats.

Methods: Six groups of female SD rats (n = 13) were administereddrinking water containing 0%, 0.5%, and 1.5% GTPs (wt/vol) for 6 mo.At each treatment level, one group of rats was killed after 3-month, andthe other rats were killed after 6 mo. Colorectal contents were collectedfor HPLC and GC-MS metabolomic analysis.

Results: GC-MS metabolomics captured 2668 features. A totalof 57 metabolites with remarkable change were imputatively identi-fied based on fragment spectra documented in the NIST database.Principal-component analysis (PCA) found that nearly 90% of datavariance was explained with PC1 and PC2. Two-way ANOVAshowed that GTP administration resulted in dose-dependent changesof 53 metabolites, time-dependent changes of 14 metabolites, anddose × time–dependent changes of 39 metabolites. Pathway analysisshowed that GTPs enhanced gut-microbiota–dependent metabolism ofcalorific carbohydrates and amino acids, elevated vitamin productionof gut microbiota, reduced absorption of fats, and reduced levelsof bile acids and cholesterol in gut. A group of key metaboliteswas quantitated and elevated levels were found for niacin (8.61-fold), 3-phenyllactic acid (2.20-fold), galactose (3.13-fold), mannose(2.05-fold), pentadecanoic acid (2.15-fold), and lactic acid (2.70-fold); reduced levels were found for cholesterol (0.29-fold), cholicacid (0.62-fold), deoxycholic acid (0.41-fold), trehalose (0.14-fold),glucose (0.46-fold), fructose (0.12-fold), alanine (0.61-fold), and proline(0.11-fold).

Conclusions: Our metabolomics findings are in line with the alter-ations of gut-microbiota community structure and enzyme orthologspreviously revealed by microbiome and metagenomic analysis. Thesefindings suggested that GTPs can effectively modify gut-microbiota–dependent energy and micronutrient metabolism in rats.

Funding SourcesUSAID grant to fund global UGA peanut research (grant ECG-

A-00-07-00001–00); the National Institutes of Health Fogarty Inter-national Center (grant1R24TW009489), and the National Center forComplementary and Integrative Health (grantU01AT006691).

Effects of Tocotrienol and Statin Supplementation on Micro-biome and Glucose Homeostasis in Inbred Obese Mice (OR34-03)

Chwan-Li Shen,1 Umesh Wankhade,2 Sree Chintapalli,2 BrianD Piccolo,2 Michael D Tomison,1 Kandis Wright,1 RebeccaGabrilska,1 Gurvinder Kaur,1 Jannette Dufour,1 and KartikShankar2

1Texas Tech University Health Sciences Center; and 2ArkansasChildren’s Nutrition Center

Objectives:We previously demonstrated a positive synergistic effectof tocotrienols (TTs) and statin on improving glucose homeostasis inoutbred CD-1 obese mice. This study examined the effects of TTsin conjunction with statin on microbiome profiles in inbred obese

C57BL/6Jmice.We also evaluated the effects of TT and statin on glucosehomeostasis and insulin resistance in study animals.

Methods: Forty male C57BL/6J mice were assigned to 4 groups(a 2 × 2 factorial design): high-fat diet (HFD, 58% energy fromfat), HFD + TT (TT at 400 mg/kg diet), HFD + statin (lovastatinat 120 mg/kg diet), and HFD + TT + statin for 14 wk. Bodyweight and food intake were measured weekly and did not differbetween groups. Glucose tolerance (GTT) and insulin tolerance (ITT)tests were performed. After 14 wk, cecal contents were collected forfurther analysis. Microbial community profiling was conducted withthe use of amplicon sequencing of the 16S rRNA gene (V4 region)followed by analysis with QIIME 1.91 and standardized pipelines inR. Data were analyzed by two-way ANOVA followed by post-hocanalysis.

Results: TT had no impact on α-diversity measures in mice. Statin-supplemented mice showed less α-diversity [at operational taxonomicunit (OTU), class, order, family, and genus levels] than those withoutstatin supplementation (P < 0.05). Principal-coordinate analysis ofβ-diversity values showed significant effects of both TT (at OTUlevel) and statin (at class, family, genus, and OTU levels) (P < 0.05).Both TT and statin affected abundance of 2 phyla and 7 genera inobese mice (P < 0.05). TT increased abundance of Acitnobacteria andsuppressed Firmicutes phylum in obese mice, whereas statin decreasedActinobacteria phylum but increased Verrucomicrobia. TT increasedabundance of SMB53 and decreased Anaerostipes genus, whereas statindecreased Lactobacillus and Anaerostipes and increased Dorea andOscillospira in obese mice. There was a significant interaction betweenTT and statin in GTT (P = 0.024) and statin in ITT (P < 0.001) inobese mice. The HFD + statin group had the lowest GTT level. Theorder of ITT was HFD = HFD + TT > HFD + TT + statin > HFD +statin.

Conclusions: Our data that demonstrate TT and statin collectivelyaltered microbial ecology in obese mice. Such changes in microbialcommunity may, in part, be associated with modulating the glucosehomeostasis and insulin resistance of the host, modulation of other hostsystems, and direct effects of the compounds on the bacteria resident inthe gut.

Funding SourcesFunded by American River Nutrition, Inc., Hadley, MA, and in part

by USDA-ARS Project 6026-51000-010-05S.

Impact of Milk Polar Lipids on High-Fat–Induced Body WeightGain, Bifidobacteria, and Gut Barrier Markers in Mice: Role ofSphingomyelin In Vitro (OR34–04)

Marine Milard,1 Fabienne Laugerette,1,2 Armelle Penhoat, 1,2

Emmanuelle Meugnier, 1,2 Emmanuelle Loizon, 1,2 Annie Du-rand, 1,2 Charline Buisson, 1,2 Audrey Neyrinck,3 NathalieDelzenne,3 and Marie-Caroline Michalski1,2

1Université Claude Bernard, Lyon, France; 2Université Lyon, Car-MeN Laboratory, INSERM, INRA, INSA, Lyon, France; and 3LouvainDrug Research Institute, Metabolism and Nutrition Research Group,Université catholique de Louvain, Brussels, Belgium

Objectives: Obesity onset is associated with alterations of the gutfunction and microbiota. Interest is growing in the metabolic impact of


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milk polar lipids (MPLs), which are rich in sphingomyelin (SM, 25% ofMPL), notably regarding colon cancer prevention and lipid metabolismimprovement in rodentmodels. Milk SMwas also reported tomodulategutmicrobiota inmice.We therefore tested the following hypotheses: 1)in mice, addition of MPLs to a high-fat diet can improve microbiotaprofile and gut barrier components including tight junction proteins(TJPs); and 2) in vitro, milk SM can participate in the modulation ofTJP expression.

Methods: Male C57Bl/6J mice (8 wk old) were fed experimentaldiets for 8 wk: high-fat devoid of MPLs (HF, 21% fat, mainly palmoil, in chow), HF-1.1% MPL or HF-1.6% MPL (HF supplementedwith 1.1% or 1.6% MPLs); a chow diet was used as a reference. Weanalyzed biometric parameters, histomorphology of the gut (cryptdepth, expression of TJPs involved in paracellular permeability), andbacteria of the caecal content (16S rDNA). In vitro, Caco-2 cells wereincubated with mixed lipid micelles devoid of or enriched with 0.2 or0.4 mMmilk SM; TJPs were analyzed by polymerase chain reaction andimmunofluorescence.

Results: The supplementation with 1.6% MPL prevented HF-diet–induced body weight gain (P < 0.01 compared with HF). In cecalmicrobiota, HF-1.1% MPL induced a specific increase in Bifidobac-terium (P < 0.05 compared with HF), in particular Bifidobacteriumanimalis. HF-1.6% MPL induced a specific decrease in Lactobacteriareuteri compared with HF (P < 0.05). Markers of the gut barrier wereimproved by MPL supplementation: colonic crypt depths were greatestwith HF-1.6% MPL (P < 0.05 compared with HF-1.1% MPL). MPLincreased the gene expression of ZO-1 in the duodenum (HF-1.6%MPLcompared with HF: P < 0.07). In Caco-2 cells, 0.4 mM of milk SMincreased the gene expression of TJPs (ZO-1, occludin, JAM-1, claudin-1; P < 0.05 compared with micelles devoid of SM) and the proteinexpression of occludin.

Conclusion: Our results show that MPLs can limit HF-inducedbody weight gain, modulate the abundance of beneficial bacteria of thegut microbiota, and stimulate the expression of TJPs in the intestine,possibly by a specific action of milk SM. This can contribute to explainreported beneficial effects of MPLs in mice regarding HF-inducedmetabolic disorders.

Funding SourcesCNIEL (French Dairy Interbranch Organization).

Dietary Whole Strawberry Inhibited Colonic Inflammationin Dextran Sulfate Sodium–Treated Mice via Restoring ImmuneHomeostasis and Alleviating Gut Microbiota Dysbiosis (OR34-05)

Yanhui Han,1 Min Gu,1 Mingyue Song,2 Daoyuan Ren,3 Xiao-qiong Cao,1 Xiaoai Zhu,1 Fang Li,1 Weicang Wang,1 Biao Yuan,4Xiaokun Cai,1 Tim Goulette,1 and Hang Xiao1

1University of Massachusetts, Amherst; 2South China AgriculturalUniversity; 3Shannxi Normal University, China; and 4Nanjing Agricul-tural University, China

Objective: Strawberry (Fragaria chiloensis) is a major edible berrywith various potential health benefits. However, detailed mechanis-tic information on the biological effects of strawberry is lacking,which greatly limits its utilization for health promotion. This studydetermined the protective effects of dietary intake of whole straw-berry (WS) against dextran sulfate sodium (DSS)–induced colitis inmice.

Methods: Sixty male CD-1 mice were randomly assigned tothree noncolitic groups and three colitic groups. Colitis was in-duced by 4 cycles of DSS (1.5% in the drinking water) treat-ments (4 d/cycle, with a 7-d recovery period after each of first3 DSS cycles), whereas noncolitic mice were supplied with reg-ular drinking water. Both colitic and noncolitic mice were givenAIN93G diet with or without 2.5% or 5.0% (wt/wt) freeze-driedWS .

Results: Our results demonstrated that dietary WS reduced thedisease activity index, prevented colon shortening and spleen enlarge-ment, and alleviated histologic damages to colonic tissues in the coliticmice. The abundance of proinflammatory immune cells was reducedby dietary WS in the colonic mucosa, which was accompanied by thesuppression of abnormal overproduction of proinflammatory cytokinessuch as tumor necrosis factor α, interleukin 1β , and interferon γ inthe colon of the colitic mice. Western blotting and immunohistochemicanalysis revealed that dietary WS decreased the expression levels ofproinflammatory proteins in the colonic mucosa. Moreover, dietaryWS partially reversed DSS-induced alteration of gut microbiota inthe colitic mice by increasing the abundance of Lactobacillus andBifidobacterium and decreasing the abundance of Akkermansia andDorea. Dietary WS also restored the decreased production of short-chain fatty acids in the cecum of the colitic mice. The changes inthe gut microbiota induced by dietary WS were closely correlatedwith its effects on proinflammatory cytokines and short-chain fattyacids.

Conclusions: Our results demonstrated the protective effectof dietary WS against the development of colonic inflammationin DSS-treated mice. This protective effect was closely associ-ated with restoration of immune homeostasis by dietary WS, andits ability to alleviate the dysbiosis of gut microbiota in coliticmice.

Funding SourcesUSDA Grants on bioactive food components (2014-67021,

MAS00450 and MAS00492).



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The Effect of DietaryAgaricus bisporusMushroomConsumptionin Pigs on Intestinal Microbiota Composition and Host Immuno-logic Function (OR34–06)

Gloria I Solano-Aguilar,1 Saebyeol Jang,1 Sukla Lakshman,1Richi Gupta,2 Aleksey Molokin,1 Ethiopia Beshah,1 MasoumehSikaroodi,2 PatrickGillevet,2 BryanVinyard,1 and JosephUrban1

1USDA-ARS; and 2George Mason University, VA

Objective: Edible mushrooms have been suggested to be potentialprebiotics. This study was designed to determine if diet supplementedwith mushroom can affect immune response and modulate intestinalmicrobiota composition and function in pigs.

Method: Thirty-one 6-wk-old pigs were randomly assigned byweight into 3 experimental treatment groups (n = 10–11/group)fed a pig growth diet alone (21% , 65%, and 15% energy fromprotein, carbohydrate, and fat, respectively) or supplemented for 6 wkwith either 3 or 6 servings of freeze-dried white button-mushroompowder (1 serving = 75g of fresh mushroom). Peripheral bloodmononuclear cells (PBMC) and alveolarmacrophages (AMs) isolated atthe end of the treatment were stimulated with Salmonella typhymuriumlipopolysaccharide (LPS). DNA of fecal and proximal colon contentswas isolated and used for microbiota composition analysis of bacterial16S rDNA. Linear discriminant analysis effect size was used todetermine relative enrichment or depletion in bacterial taxa in response


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to diet. Operational taxonomic units (OTUs) were normalized fornumber of 16S rDNA gene copies, and used to predict metagenomefunctional content through the use of the Phylogenetic Investigation ofCommunities by Reconstruction of Unobserved States (PICRUSt).

Results: Pigs gained weight over the 6 wk with no differencein body composition, bone density, or intestinal permeability. Theconsumption of 3 or 6 servings of mushroom significantly reducedLPS-induced interleukin (IL)-1β gene expression in AMs (P < 0.05),whereas consumption of 6 servings of mushroom decreased LPS-induced gene expression of IL-8 and IL-10 ( P< 0.01) with no change intumor necrosis factor α or IL-6 in AMs or in LPS-stimulated PBMCs.Analysis of the 16S rDNA gene in fecal samples indicated changes infecal microbiota structure, with an increase in bacterial families in theorder Clostridiales and a shift in host bacterial function with increasedcarbohydrate metabolism and biosynthesis of secondary metaboliteswith mushroom supplementation.

Conclusion: Consumption of dietary mushroom induced changesin host microbiota composition with increased carbohydratemetabolism and modulation of host immunity by reducinginflammatory responses in AMs and PBMCs that promote health.

Funding SourcesUSDA ARS and the Mushroom Council.

Structurally Different Oat Ingredients Stimulate Specific Micro-bial Pathways in the Gut Microbiome of Three Human IndividualsIn Vitro (OR34-07)

Alison Kamil,1 Pieter Van den Abbeele,2 Lisa Fleige,1 YongsooChung,1 Peter De Chavez,1 and Massimo Marzorati3

1PepsiCo, Inc.; 2 ProDigest bvba; and 3Center of Microbial Ecologyand Technology, Ghent University, Belgium

Objectives: Preliminary evidence, primarily in animal and in vitrostudies, suggests that oats have prebiotic potential. Prebiotics areingredients that are selectively utilized by host microorganisms andconfer a health benefit. Prebiotics tend to be selectively fermentedmost by bifidobacteria. We conducted an in vitro screening of 6 oatingredients (Old Fashioned, Instant, Oat Bran, Steel Cut, Pre-CookedOat Flour, and Pre-Cooked Morrison Oat Flour) for prebiotic activitycompared with fructo-oligosaccharide (FOS)-positive control and ablank negative control.

Methods: Oat prebiotic activity was evaluated by batch colonicfermentation with the use of sugar-depleted nutritional mediuminoculated with fecal material of 3 independent human donors. Tosimulate in vivo conditions, 4 of the 6 oat ingredients were cooked (OldFashioned, Instant, Oat Bran, and Steel Cut). Prior to fermentation, weperformed in vitro digestion with dialysis to simulate upper gastroin-testinal conditions (with exemption of FOS). The oat ingredients werestandardized for β-glucan (βG) and moisture content after cookingbefore dosing into the gastric and colonic suspensions. Prebiotic activitywas assessed over 0–48 h by measuring the composition of microbialcommunity (bifidobacteria, lactobacilli, firmicutes) through the use ofquantitative polymerase chain reaction andmicrobialmetabolic activity[gas production, pH, short-chain fatty acids (total, acetate, propionate,butyrate, branched), ammonium, lactate].

Results:All oat ingredients statistically increased levels of bifidobac-teria compared with negative control but there were no significantchanges in lactobacilli. All oat ingredients statistically increased levelsof total short-chain fatty acids and propionate compared with negativecontrol. Propionate production was higher for all oat ingredientscompared with FOS but not for other endpoints. There were nostatistically significant differences between oat ingredients for any of theanalyses.

Conclusions: All oat ingredients, standardized for βG content,exhibited some prebiotic activity compared with negative control. Alloat ingredients statistically increased bifidobacteria levels. However,none of the oat ingredients reached the overall levels observedwith FOS.The ability to replicate these results in vivo may be dependent on βGamount.

Funding SourcesPepsiCo, Inc. financially supported the study.

Cranberry Constituents Abolish the Impact of a Plant Food–Free Diet on Microbiota Composition and Carcinogenic Bile Acids(OR34-08)

Jose Rodriguez-Morato,1,2 Nirupa Matthan,2 Rafael de la Torre,3and C-Y Oliver Chen2

1Universitat Pompeu Fabra, Spain; JeanMayer USDAHNRCA,MA;and 3Hospital del Mar Medical Research Institute, Spain

Objective: Foods and gut microbiota modulate human healththrough their reciprocal interactions. Cranberries have multiple healtheffects but the impact of their constituents on the gut microbiotahas not been examined in humans. Thus, the aim of this study wasto evaluate the reciprocal relationship between the microbiota andcranberry constituents in the context of a plant food–free diet.

Methods: We conducted a randomized, crossover, placebo-controlled feeding trial with two 5-d intervention phases and a 2-wkwashout period. Healthy adults (n= 11, aged 39 y, bodymass index 22.2kg/m2) were randomly assigned to receive either control diet [(CON)plant food–free diet plus 30 g/d placebo powder] or cranberry diet[(CRA) freeze-dried whole cranberry powder to replace the placebo].The plant food–free diet comprised only meats, dairy products, andsimple sugars. Samples were obtained before and after each interventionphase for the analysis of fecal microbiome, bile acids, short-chain fattyacids (SCFAs), and trimethylamine (TMA), urinary anthocyanins andphenolics, and plasma cytokines.

Results: Linear discriminant analysis effect size (LEfSe) showedthat, compared with habitual diet, CON modified the abundanceof 46 taxonomic clades with a linear discriminant analysis score>2, i.e., Firmicutes, Bacteroidetes, Clostridiales, Clostridia, Anaerosipes,Lachnospira, S. moorei, R. gnavus, and R. bromii. Moreover, CONincreased bacteria-derived deoxycholic acid by 268%, and decreasedacetate and butyrate by 40% and 48%, respectively (P ≤ 0.05). Ascompared with the postintervention phase of CON, LEfSe showed thatCRA modified the abundance of 9 taxonomic clades, i.e., Firmicutes,Bacteroidetes, Anaerostipes, Lachnospira, and Oribacterium. Further,CRA abolished CON-induced increase in deoxycholic acid (P ≤ 0.05)and increased urinary anthocyanins and 3,4-dihydroxyphenylacetic


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and homovanillic acid (P ≤ 0.05). No changes were found in TMA andcytokines.

Conclusions: A plant food–free diet could alter the microbiotacomposition to a less favorable profile, increase deoxycholic acid, anddecrease beneficial SCFAs. Cranberry constituents abolished the impactof the plant food–free diet on microbiota composition and bile acids,suggesting health benefits of cranberries via modulation of the gutmicrobiota.

Funding SourcesThis research was funded by the Cranberry Institute and USDA.

JRM acknowledges funding from European Union’s Horizon 2020research and innovation programme under the Marie Skłodowska-Curie grant agreement 712949 (TECNIOspring PLUS) and fromACCIO.

Curcumin and Piperine Repress Intestinal mTORC1-DependentExpression of Proinflammatory Cytokine Tumor Necrosis Factor α

(OR38-01)

Harleen Kaur, Bo He, and Regis Moreau

University of Nebraska-Lincoln

Background: Persistent activation of the mechanistic target ofrapamycin complex 1 (mTORC1) is linked to sustained inflammationand progression of colorectal cancer. Curcumin, a bioactive polyphenolpresent in turmeric, is reported to have anti-inflammatory and anticar-cinogenic activities. However, curcumin’s bioavailability is limited dueto active metabolism in the intestines and luminal export. Piperine,an alkaloid present in black pepper, can increase the bioavailabilityof curcumin by inhibiting intestinal and hepatic biotransformationenzymes.

Objective: The aim of this study was to determine whethercurcumin and piperine have individual and combined effects inmitigating gut inflammation via regulation of mTORC1 in humanintestinal epithelial cells.

Methods: HT-29 and Caco-2 cells were treated with curcumin andpiperine either alone and in combination to determine their effectson mTORC1 activity, protein synthesis, and tumor necrosis factor α

(TNFα) expression.Results: Curcumin repressed mTORC1 activity (measured as

changes in the phosphorylation state of its downstream targets, p70ribosomal protein S6 kinase B1 and 40S ribosomal protein S6) ina dose-dependent manner (2.5–20 μM, P < 0.007), and suppressedthe synthesis of nascent proteins. Piperine inhibited mTORC1 activity,albeit at comparatively higher concentrations than curcumin. Thecombination of curcumin + piperine further repressed mTORC1signaling (P < 0.02). Mechanistically, curcumin represses mTORC1by preventing TSC2 degradation, the conserved inhibitor of mTORC1in HT-29 cells (P < 0.05). The mode of action of curcumin wasdifferent in HT-29 compared with Caco-2 cells. In Caco-2 cells,curcumin repressed mTORC1 activity by lowering the phosphorylationstate of PRAS40 (Thr246, P < 0.05), which inhibits mTORC1 whendephosphorylated. Furthermore, we show that a functional mTORC1is required for the transcription of TNFα as Raptor knockdownabrogated TNFα gene expression. Curcumin, piperine, and theircombination inhibited mTORC1-mediated TNFα gene expression,

but failed to do so when TSC2 was knocked down in HT-29cells.

Conclusion: Curcumin and piperine, either alone or in combina-tion, have the potential to downregulate mTORC1 signaling in theintestinal epithelium, a finding that has implications for tumorigenesisand inflammatory disease.

Funding SourcesUSDA-NIFA and USDA Hatch Act.

Blackcurrant (Ribes nigrum) Exerts an Anti-Inflammatory Ac-tion by Modulating Macrophage Phenotypes (OR38-02)

Yoojin Lee, Tho Pham, and Ji-Young Lee


Objectives: Based on our previous finding of anti-inflammatoryeffects of blackcurrant, we investigated if blackcurrant exerts anti-inflammatory actions via modulation of macrophage phenotypes.

Methods: Seven-week old male C57BL/6J mice were fed either ahigh-fat/high-sucrose control diet (HF/HS) or a HF/HS diet containing6%blackcurrant (HF/HS-BC) for 26wk. Splenocytes isolated frommicefed an HF/HS or HF/HS-BC diet were stimulated with 500 ng/mL oflipopolysaccharides (LPS) for 3 h ex vivo. RAW 264.7 macrophageswere pretreated with 0, 25, 50, 75, and 100 μg/mL of polyphenol-richblackcurrant extract (BCE) for 24 h, and then stimulated with 100ng/mL of LPS for 6 h. Bone marrow cells from C57BL/6J mice weredifferentiated into macrophages (bone-marrow-derived macrophages,BMDMs) for 7 d. BMDMs were then polarized into M1 macrophagesby interferon-γ and tumor necrosis factor α (TNFα) (10 ng/mLeach) or M2 by interleukin (IL)-4 and IL-13 (10 ng/mL each) in theabsence or presence of 50 μg/mL of BCE for 24 h. A set of polarizedBMDMs were stimulated with 10 ng/mL of LPS for an additional6 h. mRNA levels of M1 and M2 markers and proinflammatorygenes were determined by quantitative real-time polymerase chainreaction.

Results: Splenocytes from BCE-fed mice showed decreased mRNAlevels of I1-1β and Tnfα, upon LPS challenge compared with thesplenocytes from control mice. BCE also reduced the expression of I1-1β and Il-6, but not Tnfα, in RAW 264.7 macrophages. Furthermore,we observed an inhibitory action of BCE during polarization ofBMDMs into M1 or M2 macrophages. Polarization of BMDMs intoM1 macrophages induced the expression of I1–1β , Tnfα, Cxcl9, andNos2; however, BCE attenuated the induction of I1-1β , Cxcl9, andNos2. Similarly, BCE attenuated mRNA levels of arginase1, clusterof differentiation 206, Ym1, and resistin-like molecule α when itwas present during M2 polarization. Additionally, LPS stimulation inpolarized BMDMs increasedmRNA abundance of I1-1β , Il-6, and Tnfαin bothM1 andM2macrophages, whichwas reduced by BCE regardlessof macrophage phenotypes.

Conclusions: Blackcurrant showed anti-inflammatory effects exvivo and in vitro. This effect is likely attributable, at least in part,to the modulation of macrophage polarization by blackcurrant, itspolyphenols in particular.

Funding SourcesUSDA AFRI (2016-67017-24463); USDA Hatch CONS00972;

USDAMulti-State Hatch CONS00916.


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Oat Avenanthramides Protect against Eccentric Exercise–Induced Muscle Inflammation in Humans after Downhill Running(OR38-03)

Tianou Zhang,1 Yuzi Zhang,1 Tao Liu,1 Gilles Gagnon,2 Jacque-line Ebrahim,2 Dongwook Yeo,1 and Li Li Ji1

1University of Minnesota; and 2Ceapro Inc.

Objectives: Avenanthramides (AVAs) are a group of diphenolicacids, found only in oats, that provide antioxidant protection and inhibitinflammation. Downhill running (DR), a typical type of eccentricexercise, activates a series of inflammatory responses in skeletalmuscles,exhibited as painful, red, and warm swelling. The objective of the studywas to evaluate whether dietary AVA supplementation could attenuateeccentric exercise–induced muscle inflammation.

Methods: Subjects (12 male and 12 female) were assigned to high-AVA (H-AVA) or low-AVA (control) groups. Two treadmill-based DRsessions were separated by an 8-wk washout period followed by 8-wk oat AVA supplementation through receiving 2 cookies/d. Bloodsamples were collected before DR and at various time points (0, 4,24, 48, 72 h) after DR. Circulatory inflammatory cytokines interleukin(IL)-6 and IL-1Receptor antagonist (IL-1Ra) and chemokinesmonocytechemotactic protein (MCP)-1 and vascular cell adhesion molecule(VCAM)-1 were measured with Luminex multiplex assays. Data wereshown as mean ± SEM and analyzed by repeated-measures ANOVA.

Results: Pilot results showed that DR significantly increased IL-6, VCAM-1, and MCP-1 levels at post-DR and 4 h post-DR groups(P< 0.01). Interaction effects were found between oat supplementationand time for MCP-1 (P < 0.05), as well as between AVA dosageand time for VCAM-1 (P < 0.05). Oat supplementation decreasedMCP-1 (P = 0.187) and VCAM-1 (0.05 < P < 0.1) at 24 h post-DRregardless of AVA treatment. Inflammatory cytokine IL-6 decreased inthe H-AVA group compared with the control group at 4 h post-DR,but no statistically significant difference was found (P > 0.05). Anti-inflammatory cytokine IL-1Ra showed an increasing trend in H-AVAgroups compared with control groups at post-DR (P = 0.158) and 48 hpost-DR (P = 0.139).

Conclusions: Based on the preliminary data, we conclude thatoat AVA supplementation could reduce circulatory inflammation, andinhibit expressions of chemokines and adhesion molecules.

Funding SourcesPepsiCo and Ceapro Inc.

Polysaccharides from Pleurotus eryngii Inhibit theLipopolysaccharide-Induced Inflammatory Response in RAW264.7Macrophages through the Mitogen-Activated Protein Kinase andNuclear Transcription Factor κB Pathways (OR38–04)

Gaoxing Ma,1,2 Liyan Zhao,1 Anxiang Su,1 Lei Zhong,1 QiuhuiHu,1 and Hang Xiao2

1Nanjing Agricultural University, China; and 2University of Mas-sachusetts Amherst

Objectives: Edible mushroom polysaccharides exhibit potential inmodulating various biological processes. In the present study, the anti-inflammatory effects of two novel water-soluble polysaccharides fromthe edible mushroom Pleurotus eryngii were investigated.

Methods: Two novel polysaccharides, PPEP-1 and PPEP-2, wereisolated and purified from the mushroom by DEAE cellulose-52chromatography and Sephadex G-200 size-exclusion chromatogra-phy. Molecular weights and monosaccharide analysis were deter-mined by high-performance size-exclusion chromatography and GC,respectively. Fourier transform infrared spectrometry (FT-IR) wasapplied to analyze the functional groups of the 2 polysaccha-rides. Finally, the anti-inflammatory effects of PPEP-1 and PPEP-2 were revealed in lipopolysaccharide (LPS)-stimulated RAW264.7macrophages.

Results: The molecular weights of PPEP-1 and PPEP-2 were foundto be 167 and 274 kDa, respectively. GC analysis indicated that PPEP-1 and PPEP-2 were heterpolysaccharides and mainly composed ofglucose. FT-IR revealed that both PPEP-1 and PPEP-2 were mainlyconsisted of β-type glycosidic linkages. Direct exposure to PPEP-1 and PPEP-2 significantly inhibited the LPS-induced inflammatoryresponses of RAW 264.7 macrophages by inhibiting the productionof nitric oxide (NO), prostaglandin E2, interleukin (IL)-1β , tumornecrosis factor α and IL-6. PPEP-1 and PPEP-2 inhibited the ac-tivation of mitogen-activated protein kinase pathways by inhibitingphosphorylation of p38, extracellular regulated protein kinases 1/2, andstress-activated protein kinase/jun aminoterminal kinase. Moreover,PPEP-1 and PPEP-2 also inhibited nuclear transcription factor κBsignaling by reducing the nuclear translocation and phosphorylationof p65.

Conclusions:Overall, our results demonstrated for the first time theanti-inflammatory effects of 2 novel polysaccharides from the ediblemushroom Pleurotus eryngii, and highlighted the potential benefits ofusing the polysaccharides as anti-inflammatory agents in functionalfoods or dietary supplements to combat inflammatory diseases.

Funding SourcesThis work was supported by China Agriculture Research System

(CARS-20).

γ-Tocotrienol Attenuates the Aberrant Lipid Mediator Produc-tion in NLRP3-Inflammasome-Stimulated Macrophages (OR38-05)

Yongeun Kim,1 Mark Brown,2 andSoonkyu Chung3

1Nutrition and Health Sciences; 2Cleveland Clinic, OH; 3Universityof Nebraska-Lincoln

Objectives: The activation of Nod-like receptor protein 3 (NLRP3)inflammasome in innate immune cells accompanies a massive pro-duction of proinflammatory lipid mediators, eicosanoids, which playa crucial role in propagating inflammation. γ -Tocotrienol (gT3) is anunsaturated vitamin E that has been demonstrated to attenuate NLRP3inflammasome. However, the role of gT3 in regulating eicosanoidformation is unknown. This study aims to determine the impact of gT3onmacrophage lipid profile and on the biosynthesis of proinflammatorylipid mediators.

Methods:Mass spectrometry–based shotgun lipidomic approacheswere used for lipidome analysis. For NLRP3 inflammasome activation,lipopolysaccharide-primed bone marrow–derived macrophages(BMDMs) were stimulated with saturated fatty acids (SFAs) in thepresence of gT3 (1 μM). The release of arachidonic acid (C20:4, AA)was measured by pulse-chase experiment with radiolabeled [3H]-AA,


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and secretion of prostaglandin E2 (PGE2) was measured by ELISA.The gene expression levels in regulating eicosanoids and ceramideswere quantified by polymerase chain reaction. The production ofATP from respiration and glycolysis was determined with a SeahorseXF-extracellular flux analyzer.

Results: The SFA-mediated inflammasome activation inducedrobust changes in lipid species of glycerolipids, glycerophospholipids,and sphingolipids in BMDMs, which were distinctly different in thegT3-treated BMDMs. The gT3 treatment caused substantial decreasesof lysophospholipids, diacylglycerol, and free AA, indicating thatgT3 limits the availability of AA, the precursors for eicosanoids.This was confirmed by decreased [3H]-AA release and diminishedprostaglandin E2 secretion. Concurrently, gT3 inhibited inductionof TLR2- and TLR4-induced cyclooxygenases 2 (Cox2), further sup-pressing prostaglandin synthesis. In addition, gT3 attenuated ceramidesynthesis via transcriptional downregulation of key enzymes forceramide de novo synthesis. The altered lipid metabolism duringinflammation reduced ATP production, which was partly rescued bygT3.

Conclusion:Our work revealed that gT3 induces distinct modifica-tion of macrophage lipidomes to reduces AA release and correspondinglipid mediator synthesis, leading to attenuated metabolic derangementagainst lipotoxicity.

Funding SourcesThis studywas supported byAmericanHeartAssociation SDGgrant

to SC (13SDG14410043), and a USDA-Hatch grant to the University ofNebraska-Lincoln.

AntiarthriticAction ofBlueberryPolyphenols inRabbit Synovio-cytes (OR38-06)

Sanique South, Jacquelynn Lucero, Victorine Imrhan, ChandanPrasad, Parakat Vijayagopal, and Shanil Juma

Texas Woman’s University

Objectives: The purpose of this study was to examine the an-tiarthritic effects of blueberry polyphenols (BBPs) in an in vitro modelof rheumatoid arthritis with the use of HIG-82 rabbit synoviocytesstimulated with tumor-necrosis factor α (TNFα). Rheumatoid arthritis(RA) is a chronic inflammatory disease that is characterized by synovialhyperplasia, inflammation, and joint destruction.We hypothesized thattreatment with BBPs will attenuate proliferation and modulate proin-flammatorymediators of TNFα-stimulatedHIG-82 rabbit synoviocytesassociated with progression of RA.

Methods: Rabbit synoviocytes were cultured in Ham’s F-12 nutrientmix medium supplemented with penicillin-streptomycin and 10% fetalbovine serum, and incubated at 37°Cwith 5%CO2. Upon attainment ofexperimental conditions, the synoviocytes were pretreated with variousdoses of BBPs prior to stimulation with the inflammatory agent TNFα.Twenty-four hours after stimulation cell proliferation, inflammatorymediators and caspase 3 and 7 activation were analyzed.

Results: Stimulation of the HIG-82 rabbit synoviocytes with theproinflammatory cytokine TNFα increased cell proliferation by ∼19%compared with the nonstimulated control. However, cell proliferationwas significantly decreased in a dose-dependentmanner with treatmentwith BBPs. Reverse transcription polymerase chain reaction analysis

showed matrix metalloproteinase 3 increased 5-fold in the controlTNFα-stimulated group but was decreased by 3-fold at a dosage of 200µg/mL in the treatment group. Similar results were exhibited from in-cell ELISA assessing inflammatory mediators. There was a decrease inC-reactive protein, and cyclooxygenase 2 expression in cells treatedwithBBPs compared with control TNFα-stimulated cells.

Conclusions: Increased proliferation of synovial fibroblasts has beenimplicated in the initiation and progression of RA. BBPs exhibitedantiarthritic abilities through decreased proliferation and attenuation ofproinflammatorymediators of synoviocytes. The antiarthritic actions ofBBPs may be of value as a therapeutic strategy in RA.

Inhibitory Effects of Astaxanthin onHelicobacter pylori–inducedMitochondrial Dysfunction and Interleukin 8 Expression in GastricEpithelial Cells (OR38-07)

Suhn Hyung Kim, Joo Weon Lim, and Hyeyoung Kim

Yonsei University, Japan

Objectives: Helicobacter pylori infection is a risk factor for gastritis.In several cell lines, mitochondrial reactive oxygen species (ROS) actas a signaling molecule to trigger inflammatory cytokine production.Astaxanthin (AST) is an antioxidant known to protect cells against ROSproduction in vivo and in vitro. The present study was undertakento determine whether H. pylori induces mitochondrial dysfunctionand mitochondrial ROS-mediated interleukin (IL)-8 expression, andwhether AST inhibits mitochondrial dysfunction and IL-8 expressionin gastric epithelial cells infected with H. pylori.

Methods: Human gastric epithelial AGS cells were infected withH. pylori (NCTC11637) at a bacterium/cell ratio of 50:1. Intracellularand mitochondrial ROS were measured with DCF-DA and MitoSoxfluorescence, respectively. Mitochondrial membrane potential (MMP)and ATP level was measured by flow cytometric analysis and kit assay,respectively, in order to determine mitochondrial dysfunction. IL-8 mRNA expression was determined by real-time polymerase chainreaction analysis.

Results: H. pylori induced intracellular and mitochondrial ROSproduction and IL-8 expression in AGS cells. H. pylori decreasedMMP and intracellular ATP level in AGS cells, indicating that H.pylori causes mitochondrial dysfunction. Apocynin, NADPH oxidaseinhibitor, inhibited H. pylori–induced mitochondrial ROS productionand IL-8 expression. The results demonstrate that NADPH oxidaseactivation mediates H. pylori–induced mitochondrial ROS productionand inflammatory signaling. AST inhibited H. pylori–induced ROSproduction, mitochondrial dysfunction, and IL-8 expression in thecells. These results suggest that AST inhibits H. pylori–induced IL-8 expression through suppressing mitochondrial ROS production ingastric epithelial cells.

Conclusions:ASTmay inhibit gastric inflammation associated withH. pylori infection by suppressing mitochondrial dysfunction and IL-8expression in gastric epithelial cells.

Wheat Germ Supplementation Promotes Gut and SystemicAntiinflammatory Milieu in C57BL/6 Mice Fed a High-Fat, High-Sucrose Diet (OR38–08)


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Babajide Ojo, Crystal O’Hara, Guadalupe Davila De El Rassi,Jerry W Ritchey, Daniel Lin, Winyoo Chowanadisai, Brenda JSmith, and Edralin Lucas

Oklahoma State University

Objective: This study investigated the effects of wheat germ (WG)supplementation in modulating changes to the normal gut bacterialpopulation and inflammatory markers in the gut and the blood of micefed a high-fat, high-sucrose (HFS) diet.

Methods: Six-week-oldmaleC57BL/6micewere randomly assignedto 4 groups (n = 12/group), and fed a control (C, 10% kcal fat) orHFS (60% kcal fat) diet with or without 10% WG (wt/wt) for 12wk. Cecal bacteria were assessed via 16S rDNA sequencing, whereasfecal short-chain fatty acids were measured by gas chromatography.Small intestinal CD4+ lymphocytes were evaluated by flow cytometry,whereas gut antimicrobial peptide genes and inflammatory markerswere also assessed by reverse transcriptase-quantitative polymerasechain reaction and ELISA, respectively. Nonmicrobiome data wereanalyzed by 2-way ANOVA with HFS and WG as factors.

Results: A 399% significant increase (P = 0.007) was observedin the abundance of the beneficial bacterial family Lactobacillaceaein the HFS + WG group compared with the HFS-fed group. WGsupplementation in the control diet raised propionic and butyricacid concentrations compared with other groups (PHFS × WG ≤ 0.01).Furthermore, T-regulatory cells (CD4+FOXP3+) in the small intestinewere significantly higher (≥168%, PWG = 0.005) in both WG-supplemented groups compared with their respective unsupplementedgroups, indicating an anti-inflammatory gut environment with WGfeeding. Similarly, WG feeding increased (PWG = 0.038) the anti-inflammatory cytokine gene, Il-10, in the ileum. Moreover, the antimi-crobial peptide genes (Reg3β andReg3γ ) were upregulated in the ileumby at least 95% (PHFS × WG ≤ 0.02) in the HFS + WG group comparedwith other groups. Interestingly, the proinflammatory cytokines tumornecrosis factor α, interleukin (IL)-1β , IL-6 and interferon γ weresignificantly lower (PWG < 0.01) in the serum of WG-supplementedmice.

Conclusion: WG showed strong gut modulatory and anti-inflammatory properties which may be vital in preventing highfat, high-sucrose diet–induced complications. Indeed, WG may pose asignificant economic value to wheat growers if its anti-inflammatoryand gut modulatory potential is further studied and harnessed.

Funding SourcesOklahoma Agricultural Experiment Station.

Antidiabetic Effect ofMangifera indica: Systematic Review (P08-001)

Sepideh Alasvand and Vivian Haley-Zitlin

Clemson University, SC

Objective:Themain aimof this studywas to provide a systematic re-view examining the antidiabetic impacts of different parts ofMangiferaindica Linn. in the management of type 2 diabetes mellitus in vitro andin vivo.Natural products have long been and continue to be an attractivesource of nutritious and pharmacologic therapeutics. One such natural

product is mangiferin (MGF), the predominant constituent of extractsof the mango plantM. indica Linn.

Methods: The databases PubMed, FSTA, Web of Science, andCINAHEL were searched for the keywords diabetes∗ OR "diabetesmellitus" OR "type 2" OR "blood glucose" OR insulin∗ OR antidiabet∗

AND (mango∗ OR "mangifera indica" OR "mangifera indica L").Articles reviewed were published in English up to January 10.

Results: Twenty-five of 436 studies met the inclusion criteria.Exclusion criteria included review articles, nonspecific mango-relatedplants, and articles that did not examine glycemic status. The articlesthat met the inclusion criteria included mangifera extracts obtainedfrom M. indica Linn. bark-stem (10.7%), peel (14.3%), flesh (17.9%),kernel (7.14%), and leaf (42.9%). Commercially available or providedmangiferin was used in 10.7% of the studies. Approximately 36% ofthe studies were in vitro and 78.6% were in vivo; 22.72% were humanstudies and 77.27% animal studies. The results confirm the antidiabeticproperties of M. indica Linn. Suggested hypoglycemic mechanismsof action included gene regulation of Glut 4, Irs 1, Pi3K, and cellcycle genes, inhibition of α-glucosidase, improved antioxidant statues,improved insulin sensitivity, and facilitated glucose uptake.

Conclusion: The mango extract, mangiferin was shown to havea potential role as a nutraceutical component for improving thehyperglycemia of type 2 diabetes. At present, studies supporting anantidiabetic role for mango in humans are lacking. The efficaciousfindings in animal studies and in the human studies available indicatethat an antidiabetic role for M. indica Linn. should be pursued furtherin human research.

Funding SourcesNA.

Effect of Portfolio Functional Food Products on Lipid Manage-ment in Individuals Reluctant to Undergo Statin Therapy (P08-002)

Peter Jones,1 Soumya Alias,2 Stephanie Jew,1 Jessica V Bauman,3Elizabeth Klodas,4 and Stephen Kopecky3

1Richardson Centre for Functional Foods and Nutraceuticals,University of Manitoba, Canada; 2University of Manitoba, Canada;3Mayo Clinic, Mayo Medical Foundation; and 4Truhealth LLC

Objectives:Dietary solutions can ameliorate cardiovascular diseaserisk in statin-reluctant patients. However, no studies have beenundertaken to explore if a portfolio approach with healthy appetizingready-to-eat foods having functional food ingredients can improvecardiovascular health in this population. The present objective wasto evaluate the effect of a range of hedonically acceptable proprietaryproducts containing 4 functional bioactives on cholesterol levels instatin-reluctant participants.

Methods: A multicenter, randomized, double-blind, free-livingcrossover study composed of 2 regimented phases of 4 wk each,separated by a 4-wk washout, was conducted. Participants (n = 54)ingested 2 servings/d from an assortment of packaged, shelf-stable foodproducts as a substitute for some foods they were already consuming.Treatment products consisted of oatmeal, pancakes, cranberry bars,chocolate bars, sprinkles, and smoothies formulated specifically toprovide 5 g of fiber, 1800 mg of α-linolenic acid, 1000 mg ofphytosterols, and 1800 µmol antioxidants per serving. Control products


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were calorie-matched like items drawn from the general grocerymarketplace.

Outcome Measures: Fasting glucose, triglyceride (TG), high-density lipoprotein (HDL) cholesterol, total cholesterol (TC) concen-trations, and low-density lipoprotein (LDL) cholesterol were analyzedat baseline (day 1 and 2) and endpoint (day 29 and 30) of both phases.

Results: A reduction of 5.08% was observed in total cholesterol(P = 0.0004) with LDL cholesterol levels being reduced by 8.80%(P < 0.0001) after consumption of study foods compared with control.Circulating TG levels, HDL cholesterol, and glucose concentrationswere not influenced by study foods.

Conclusions: Consumption of a portfolio of ready-to-eat bioactivefoods significantly improves serum lipid profiles in patients unable orunwilling to take statin drugs. This novel, easily adaptable, food-basedapproach is anticipated to have extensive implications for healthcareresearch and practice improvement.

Funding SourcesTM Therapeutics, Canada and StepOne Foods, MN.

The Small Molecule Kaempferol Protects against Streptozotocin-Induced Diabetes through Suppressing Hepatic Glucose Production(P08–003)

Hana A Alkhalidy,1,2 Aihua Wang,1 Wei Zhen,1 Jing Luo,1 RyanMcMillan,1 and Dongmin Liu1

1Virginia Tech; and 2Jordan University of Science and Technology

Background: In diabetes mellitus, an increase in the activity of keyliver enzymes which control glycogenolysis and primarily gluconeo-genesis causes an increase in the rate of hepatic glucose production.This increase is the main contributor to hyperglycemia development,in particular, fasting hyperglycemia. We hypothesized that kaempferol,a naturally occurring flavonol present in some medicinal herbs andcertain types of foods, would control glucose levels in diabetes.

Objective: The aim of this study was to determine the effects of oraladministration of kaempferol in an insulin-deficient mouse model, andto explore the underlying mechanism of action of this compound.

Methods: Streptozotocin (STZ)-induced diabetic mice were orallyfed kaempferol (50 mg/kg) or a vehicle. Body weight (BW), and foodintake (FI) were recorded weekly. Nonfasting and fasting blood glucoselevels were measured biweekly. Body composition was evaluatedinitially and at weeks 4 and 8 of treatment. Pyruvate tolerance test andglucose tolerance test were performed at weeks 4 and 6, respectively.At week 12, mice were fasted and killed, and liver and muscle tissueswere analyzed for glucose oxidation and for evaluation of expressionand activity of key enzymes. Bloodwas collected for plasma lipid profile,insulin, and glucagon level determination.

Results: Oral administration of kaempferol significantly amelio-rated hyperglycemia and improved glucose tolerance. After 12 wk oftreatment with kaempferol, the occurrence of overt diabetes decreasedfrom 100% to 77.8% in STZ-induced diabetic mice. This kaempferoleffect was associated with a reduction in hepatic glucose productionand an increase in glucose oxidation in themuscle of diabetic mice withno significant changes on BW, FI, body composition, plasma insulin, orglucagon levels. On the molecular level, kaempferol treatment restoredhexokinase activity in the liver and skeletal muscle tissues, and reduced

glycogenolysis and gluconeogenesis, possibly via inhibiting pyruvatecarboxylase in the liver.

Conclusion: The findings from this study suggest that kaempferolholds a great potential to treat insulin-deficient diabetes by improvingglucose metabolism in skeletal muscle, mainly by suppressing hepaticgluconeogenesis.

Funding SourcesThe work was supported by grants from National Center for

Complementary and Integrated Health of National Institutes of Health(1R01AT007077 to DL).

Effects of DailyWhole Egg Consumption onOxidative Stress andInflammatory Markers in Healthy, Postmenopausal Women (P08–004)

Allison S Bardagjy, Jody Randolph, Lisa Sawrey-Kubicek, Cheng-hao Zhu, Romina Sacchi, Angela Zivkovic, and Francene MSteinberg

University of California, Davis

Objective: The objective of this study was to investigate the effectsof daily whole egg consumption compared with daily yolk-free eggconsumption on the antioxidant capacity of high-density lipoproteins(HDLs) and on circulatingmarkers of oxidative stress and inflammationin healthy, overweight, postmenopausal women.

Methods: This study was a 14-wk, randomized, controlled, single-blinded crossover dietary intervention trial in postmenopausal womenwith a body mass index of 25–35 kg/m2. Enrolled subjects (n = 20)were randomly assigned to consume a breakfast containing either 2whole eggs or the equivalent amount of yolk-free eggs daily for 4wk. Following a 4-wk washout, subjects continued on to the alternatedietary treatment arm. Fasting blood samples were collected beforeand after each dietary treatment to measure oxidized low-densitylipoprotein (oxLDL), paraoxonase 1 (PON1) arylesterase activity, andserum amyloid A (SAA). For statistical analysis, baseline valueswere subtracted from 4-wk values. The changes on each treatmentwere compared with the use of the Wilcoxon matched-pairs signedrank test.

Results: The median change in oxLDL was −2.1 U/L (IQR: −4.6,3.5 U/L) following whole egg treatment and 0.75 U/L (IQR: −3.3, 6.2U/L) following yolk-free egg treatment; however, these changes werenot significantly different between treatments (P = 0.31). Similarly,there were no significant effects of dietary treatment on PON1 activity,despite a significant increase in activity from baseline to 4 wk on wholeegg treatment (P = 0.014). The median change in SAA was 2.5 mg/L(IQR:−0.65, 9.4 mg/L) after whole egg treatment and−1.0 mg/L (IQR:−6.6, 4.5 mg/L) after yolk-free egg treatment; however, these changeswere not significantly different between treatments (P = 0.12).

Conclusions: Daily consumption of whole eggs compared withyolk-free eggs did not affect oxidative stress or inflammatory markersin healthy, postmenopausal women. In addition, the changes in PON1activity following each dietary treatment were not different, eventhough there was a significant increase in PON1 activity from baselineto 4 wk on the whole egg treatment. A longer intervention maybe needed to distinguish the effects of different diets on PON1activity.


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Funding SourcesAmerican Egg Board.

Effect of Green Tea Extract on Oxidative Stress and RenalFunction in Diabetic Individuals: A Randomized, Double-Blinded,Controlled Trial (P08-005)

Patrícia Borges Botelho,1 Sáskia Vaz,2 Luiza MN Amorim,2Pamella VF Nascimento,3 Valéria SP Veloso,4 Marina Nogueira,5and Inar Castro5

1Brasília University, Brazil; 2Federal University of Goiás, Brazil;3Pontifical Catholic University of Goiás, Brazil; 4Service of Nephrology,Department of Internal Medicine, Federal University of Goiás, Brazil;and 5Functional Food Development Laboratory, University of SãoPaulo, Brazil

Background and objectives: The effect of green tea on oxidativestress and renal function in diabetic patients remains inconclusive,thereby making its assessment relevant as an additional therapy forattenuation and prevention of renal disease in this population. Thus,this study aimed to evaluate the effect of green tea extract on oxidativestress and renal function in diabetic individuals.

Methods: A randomized, double-blinded, controlled clinical trialwas developed with 60 diabetic individuals. Patients were assigned to2 equal groups to receive green tea extract (n = 30, 2 capsules/d,containing 560 mg of polyphenols each) or cellulose (n = 30, 2capsules/d) for 20 wk. Blood glucose, glycosylated hemoglobin, insulin,antioxidant enzymes, total antioxidant capacity, serum creatinine, urea,glomerular filtration rate, and albuminuria were analyzed.

Results:No significant difference was observed in glycemic control,renal function, or oxidative stress, except for superoxide dismutase.After 20 wk, green tea extract maintained this enzyme activity(14.29 ± 5.00 U/mg of protein at baseline and 12.51 ± 7.39 U/mg ofprotein at 20 wk), whereas an expressive reduction in the placebo groupwas noted (13.71 ± 5.83 U/mg of protein at baseline and 8.53 ± 3.54U/mg of protein at 20 wk), with a higher superoxide dismutase activityin the green tea group after intervention (P = 0.014).

Conclusion: Green tea extract prevented the reduction of su-peroxide dismutase activity; however, it was not associated with animprovement in total antioxidant capacity, glycemic control markers, orrenal function in diabetic individuals after 20 wk of supplementation.

Funding SourcesThis research received no specific grant from any funding agency, or

commercial or not-for-profit sectors.

Montmorency Tart Cherry Supplementation and Exercise Posi-tively Affect Bone Quality in the Growing Skeleton (P08-006)

James D Bothwell, Kendall Anderson, Tiffany M Dodier, Baba-jide A Ojo, Daniel D Lin, Edralin Lucas, and Brenda J Smith

Oklahoma State University

Objectives:This study investigated the efficacy ofMontmorency tartcherry (TC) alone and in combination with exercise on improving bonequality in young growing animals and the underlying mechanisms ofaction.

Methods: Six-week-old female C57BL/6 mice were randomlyassigned to 4 groups (n = 12 mice/group) in a 2 × 2 factorial design:control AIN-93G diet (CON), CON + exercise, TC (10% wt/wt), orTC + exercise. The exercise consisted of treadmill running for 30 min,5 d/wk at 12 m/min and a 5° incline. Body weights were recordedweekly. After 8 wk of treatment, mesenchymal stem cells (MSCs) inthe tibial bone marrow were quantified via flow cytometry fluorescent-activated cell sorting. Trabecular and cortical bone microarchitecturein the femur and lumbar vertebrae was assessed by microcomputedtomography. Biomechanical testing was performed by finite-elementanalysis. The relative abundance of RNA for genes involved in osteoblastand osteoclast differentiation and activity was determined by reversetranscriptase-polymerase chain reaction.Datawere analyzed by a 2-wayANOVA with TC and exercise as factors.

Results: At the end of the study, no differences in body weight wereobserved. Trabecular bone volume in the femur and spinewas increasedwith exercise and TC (P < 0.05), but there was no interaction. Corticalbone thickness in the vertebra was also increased by TC and exercise(P< 0.001), but not in the femur. Trabecular bone strength and stiffnesswere increased in the vertebra in response to TC and exercise, butonly in response to TC in the femur (P < 0.001). An increase in bonemarrowMSCs occurred in response to exercise (P < 0.01), but not TC.However, the combination of TC and exercise reduced nuclear factorof activated T-cells 1 (Nfatc1) femur gene expression, a key regulator ofosteoclastogenesis (P < 0.05). TC also increased bone morphogeneticprotein (BMP)2 gene expression, a regulator of osteogenesis.

Conclusion: Our data indicate that TC and exercise had positiveeffects on bone quality, with effects on osteoclastogenesis and osteoge-nesis within the femur. However, the effects of TC were similar and insome cases greater than exercise.

Funding SourcesCherry Marketing Institute.

Evaluation of the Mechanism of Action of LACTIUM, a MilkHydrolysate Enriched in α-Casozepine with Anxiolytic Properties(P08–007)

Audrey Boulier,1 Nicolas Violle,2 Sylvain Denis,3 MoniqueAlric,3 Sylvain Denis,3 Monique Alric,3 Alain Baniel,1 AudreyRomelard,1 and Alain Baniel1

1Ingredia SA; 2Etap-Lab; and 3Clermont Auvergne University,France

Objective: The benzodiazepine site of GABAA receptor is a majortarget for the modulation of anxiety in both animals and humans. Theobjective is to evaluate whether flumazenil (FLU), a benzodiazepineantagonist, inhibited the anxiolytic effect of Lactium in the conditioneddefensing burying (CDB) test of anxiety in rat.

Methods: Six groups of rats were studied (n = 14). The firsttreatment was administered 80 min before testing. Each rat receivedeither FLU (10 mg/kg) or placebo (negative control) by intraperitonealinjection. Then, 60 min before testing, a second treatment of Lactium(15 mg/kg), diazepam (benzodiazepine agonist; 3 mg/kg), or placebo(negative control) was given to each of the rats by oral administration.Each rat was tested for 5 min in the CDB test, an experimental situationin which rats bury an anxiogenic probe with the sawdust present


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on the floor. For each group, the global score of anxiety (GSA) wascalculated from the measurement of the following behaviors: durationof probe-burying, number of head stretches towards the probe, numberof approach/flight sequences towards the probe. Statistical analysis wasby Kruskal-Wallis and Mann-Whitney tests.

Results: The oral administration of Lactium at 15 mg/kg 60 minbefore theCDB test induced a significant decrease in theGSAof the rats,which confirms the anxiolytic effect of Lactium. The intraperitonealadministration of FLU at 10 mg/kg 20 min before the administrationof diazepam or Lactium blocked their respective anxiolytic effects, buthad no anxiogenic effect when injected alone (Figure 1.).

This indicates that Lactium exerts its anxiolytic activity via thebenzodiazepine site of GABAA receptor, similarly to benzodiazepinessuch as diazepam.

Conclusion: The anxiolytic effect of Lactium is mainly linked tothe modulation of the GABA system via the benzodiazepine site of theGABAA receptor. The way that Lactium components reach the brainremains to be evaluated.

Funding SourcesIngredia SA.


FIGURE P08-007-1 Graph of global anxiety score.

EstimatedDietary Intake andMajor Food Sources of Polyphenolsin Mexican Adults (P08-008)

Monica L Castro Acosta,1 Ana Lopez-Palazuelos,2 PaolaGalindo-Vidales,1 Alma G Felix-Heras,1 Lorena Serrano-Corrales,1 and Marcela Vergara-Jimenez1

1Universidad Autonoma de Sinaloa, Mexico; and 2UniversidadCatolica de Culiacan, Mexico

Objective: The aim of the present study was to estimate the dietaryintake of total polyphenols and polyphenol classes, and to identify themain food sources of polyphenols in Mexican adults.

Methods:Dietary data from theMexican National Survey of Healthand Nutrition 2012 (ENSANUT 2012) were analyzed. Food-frequencyquestionnaires were collected from 3357 adults (aged18–90 y), at thenational level. Additionally, a polyphenol food composition databasewas created through the use of Phenol-Explorer and USDA databases,and extended with the use of polyphenol retention factors and food

recipes. Data are presented as median (25th and 75th percentiles), andpolyphenol intake between genders was compared by Mann-WhitneyU-test; a P-value <0.05 was considered statistically significant.

Results:Median intakes were: total polyphenols, 232 (95, 566)mg/d;flavonoids, 110 (46, 217) mg/d; phenolic acids, 49 (20, 175) mg/d;lignans, 1.76 (0.05, 5.60) mg/d; and stilbenes, 0.01 (0.00, 0.12) mg/d.Total polyphenol intake was significantly higher in the male population(n = 1366), 258 (113, 583) mg/d than in the female population(n = 1991); 211 (88, 543) mg/d, (P < 0.005). The main food sourcesof total polyphenols were: coffee (49%), fruit and vegetables (30%),and fruit juices (6%); for flavonoids: fruit and vegetables (66%), fruitjuices (13%), alcoholic beverages (8%); and for phenolic acids: coffee(82%), pastries and bread (5%), and fruits and vegetables (5%). Thefood items orange and tangerine, apple and pear, strawberries, andgrapefruit accounted for the major food sources within the fruit group.The food items green leafy vegetables, onion, carrots, and nopal (cactus)accounted for the major food sources within the vegetables group.

Conclusion: The results showed a low dietary intake of totalpolyphenols and polyphenol classes. The main food sources weresimilar to previous reports from Europe, the United States and Brazil,

where coffee, fruits, and fruit juices are themajor contributors of dietarypolyphenols. Further studies are needed to assess if the polyphenoldietary intake is related to sociodemographic and lifestyle factors in thispopulation group.

Funding SourcesUniversidad Autonoma de Sinaloa.

4-Hydroxychalcone Effect on Glucose and Lipid Profile in Ratswith Normal Diet and Simple Water Consumption as Well as Waterwith a 50% Sucrose Solution Consumption (P08-009)

Francisco Humberto Castro-Sánchez, Selene de Jesús Acosta-Cota, Dora Alicia Ochoa-Acosta, Lorenzo Ulises Osuna-Martínez, Julio Montes-Ávila, Marcela de Jesús Vergara-Jimenez

Universidad Autónoma de Sinaloa, Mexico

Objective: The aim of this study was to evaluate the effect of4-hydroxychalcone (4HC) as well as a combination of 4HC and


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metformin (Met) on the glucose and lipid profile of Wistar rats fed anormal diet and simple water (W) consumption as well as water with a50% sucrose concentration (Suc) consumption.

Methods:A total of 48Wistar rats were divided into 8 experimentalgroups (n = 6 group): 1, W + SS (saline solution); 2, W + Met; 3,W+ 4HC; 4,W+ 4HC+Met; 5, Suc+ SS; 6, Suc+ 4HC; 7, Suc+Met;and 8, Suc + 4HC + Met. The doses of treatments used per day inthe groups were 200 mg Met/kg and 80 mg 4HC/kg. All the groupsreceived the same diet (Lab Diet 5012). The duration of the study was24 wk. A blood sample was collected at the end of the study after anight of fasting. Glucose (Glu), triacylglycerol (TAG), total cholesterol(TC), and high-density lipoprotein (HDL) cholesterol were assessed bycommercial kits (RANDOX). Data were analyzed by ANOVA followedby post hoc Tuckey test with P < 0.05 set as significant.

Results: There were no differences in Glu, TAG, and HDLc betweenW + SS and Suc + SS; TC was statistically lower in Suc + SS(P < 0.05). In the groups with W there were no differences betweentreatments in Glu, TAG and HDLc as compared with W + SS. Therewere no statistically significant differences in TC between W + SS andW + Met + 4HC. The treatments with Met and 4HC separately, bothstatistically significantly decreased TC (P < 0.05). No differences wereobserved between groups with Suc in Glu and TAG; nevertheless, TCand HDLc were lower in rats with Suc + Met + 4HC when comparedwith Suc + SS (P < 0.05). Lower HDL cholesterol was also observedwith Suc + 4HC as compared with Suc + SS (P < 0.05).

Conclusion: The consumption of water with 50% sucrose concen-tration did not produce a negative effect on lipid profile and Glu.4HC decreases TC when W is consumed but when Suc is consumed,4HC + Met decreases TC but at the expense of decreasing HDLcholesterol. Due to these results, is not yet possible to suggest the useof 4HC as a treatment for lipid or glucose alterations.

Consumption of Orange Juice Associated with BalancedDiet En-hanced Vascular Endothelial Function and Reduced CardiovascularRisk Associated with Metabolic Syndrome (P08-010)

Thais B Cesar,1 Renata Oliveira,1 Olivia F Ponce,1 MichelMasser,2 and Elizabeth Baldwin3

1São Paulo State University—UNESP, Brazil; 2Federal University ofSão Carlos—UFSCar, Brazil; and 3USDA-ARS Horticultural ResearchLaboratory

Objective: The aim of this study was to investigate the effect oforange juice (OJ) along with a balanced diet on vascular endothelialfunction (VEF) and nutritional parameters in metabolic syndromepatients.

Methods: We undertook a randomized clinical study of parallelgroups with short-term dietary intervention. Subjects, aged 48 ± 9 y,were randomly assigned to Control (n= 34) andOJ (n= 34) groups. Allsubjects received nutritional counseling for a balanced diet. In addition,the OJ group also received 500 mL/d of 100% orange juice betweenmeals (twice/day), whereas the Control group consumed snacks similarin calories to OJ (noncitrus fruits or pasteurized yogurt). All patientswere followed for 12 wk, and anthropometric, dietary, biochemical,and VEF parameters, and cardiovascular risk (Framingham Risk Score)were assessed.

Results: After 12 wk of dietary intervention with or withoutOJ, significant reductions in body weight (−2%), body mass index(−3%), fat mass (−6%), waist circumference (−5%), and visceral fat(−4%) were observed, without changes in lean mass. In the OJ group,significant reductions in glycemic (−4.5%), total cholesterol (−11.6%)and low-density lipoprotein cholesterol (−17.2%) were detected. Forboth groups blood pressure decreased 8.5%, but only the OJ groupshowed a decrease of −5% in the brachial artery diameter. Thecardiovascular risk score indicated that OJ decreased by 15% the chanceof a cardiovascular event in the next 10 y, whereas the diet alonedecreased this by 12%.

Conclusion: Orange juice, coupled with a balanced diet, improvedthe vascular profile more than the balanced diet alone, with conse-quences for cardiovascular health. Both groups showed improvementin anthropometric characteristics and blood pressure.

Funding SourcesProgram for Scientific Development, Scholl of Pharmaceutical

Sciences, UNESP (PADC/FCFAr), Coordination for the Improvementof Higher Education Personnel (CAPES-Brazil), Citrosuco SA, andCitrusBR.

Orange Juice Anti-Inflammatory Effect Associated with aReduced-Calorie Diet in Obese Women (P08-011)

Thais B Cesar,1 Carolina Ribeiro,1 Renata B Oliviera,1 andElizabeth Baldwin2

1São Paulo State University—UNESP, Brazil; and 2USDA-ARSHorticultural Research Laboratory

Objective: The aim of this study was to verify the impact of orangejuice consumption on systemic low-grade inflammation parameters inobese women submitted to a reduced-calorie diet.

Methods: Fifty-four obese woman aged 33 ± 1 y with a body massindex of 35 ± 3 kg/m2 were randomly divided into 2 parallel groups:1) orange juice (n = 26): individuals submitted to a reduced-caloriediet that included orange juice (500 mL/d); and 2) control (n = 28):individuals submitted to a reduced-calorie diet without orange juice.Inflammatory parameters were analyzed over 12 wk of intervention.

Results: Regular consumption of orange juice associated witha reduced calorie diet lowered interleukin-6 by 47% (P = 0.02),ultrasensitive polymerase chain reation (us-PCR) by 42% (P = 0.001),homeostasis model assessment of insulin resistance by 34% (P= 0.001),and alanine transaminase (ALT) by 28% (P = 0.04) compared withcontrol subjects. After treatment, both groups, control and orangejuice, showed similar reductions in leptin (−16%, P = 0.40), tumornecrosis factor α (−20%, P = 0.49), alkaline phosphatase (−9%,P = 0.69), aspartate transaminase (AST) (−11%, P = 0.31), γ -glutamyl transferase (−10%, P = 0.17), and increased adiponectin(22%,P= 0.45), although these differences were not significant betweengroups. In addition, us-PCR was negatively correlated to orange juiceconsumption (−41%, P = 0.002) and positively with circulating ALT(r = 0.42, P = 0.02). Previous studies have shown that citrus flavonoidsfrom orange juice exert an anti-inflammatory effect by decreasingthe damage to hepatic and pancreatic cells, which improves insulinsensitivity and lowers liver damage. Finally, the decrease in dietary


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calories, accompanied by bodyweight loss, decreased fat percentage andwaist circumference, which was similar between groups.

Conclusions: Inclusion of orange juice in a reduced-calorie dietreduced systemic inflammation in obese women, which, in turn,benefits cardiovascular health and lessens the chance of diabetes.

Funding SourcesPADC-FCFAr/UNESP, CAPES, Citrosuco SA, CitrusBR.

Comparative Antiangiogenic Activity across Plant Anatomy:Pilot Study of Broccoli and Carrots (P08-012)

RachelAChiaverelli, Vincent Li, AbdoAbou-Slaybi, andWilliamW Li

The Angiogenesis Foundation

Objectives: Diet is an important lifestyle factor influencing cancerrisk and tumor behavior. Angiogenesis, the process of blood vesselformation, is important for tumor growth, and is a validated targetin cancer therapy. Early intervention lowers the risk of tumor de-velopment in experimental models, and correlates with populationstudies examining dietary factors. Mechanisms of angioprevenionin cruciferous and root vegetables are due to bioactives such asisothiocyanates and carotenoids. We developed an in vitro methodto quantify antiangiogenic potency in a systematic and comparativestudy of foods and beverages in the various states they are con-sumed by people. A pilot study was conducted examining broccoli(florets compared with stem) and carrots (taproot compared withleaves).

Methods: Solvent extraction was performed from natural foodsources and extracts exposed to a simulated digestive system. Broccoliflorets, broccoli stems, carrot taproots, and carrot greens were studied.Human microvascular endothelial cells (HMVECs) were culturedin growth factor–containing media and incubated with experimen-tal extracts. Cell proliferation was quantified through the use offluorescence.

Results: Compared with untreated control cells, extracts frombroccoli and carrots significantly inhibited endothelial cell proliferation(P < 0.05), at levels similar to positive control (sunitinib, an FDA-approved antiangiogenic cancer drug). There was a significant 2-fold enhancement of bioactivity when comparing broccoli stemswith florets (P < 0.05) and comparing carrot greens with taproots(P < 0.05).

Conclusions: Dietary intake of broccoli and carrots possess an-tiangiogenic bioactivity with potential to suppress tumor angiogenesis.Broccoli stem had significantly higher levels of bioactivity thanthe broccoli floret, and carrot greens had higher bioactivity thancarrot taproot, demonstrating potential health value in an ediblecomponent that is often discarded by home cooks, chefs, and the foodindustry. To our knowledge, this is the first angiogenesis study toexamine food anatomy. Our results suggest a potential sustainabilitybenefit from a broader systematic examination of foods using thisapproach.

Funding SourcesSupported byDavid Evans, AdamClayton, Jeffery Tarrant, and Flora

L Thornton Foundation.

Characterization of Differential Antiangiogenic Activity of Chi-nese, Japanese, and English Teas (P08-013)

Rachel A Chiaverelli, Vincent Li, and WilliamW Li

The Angiogenesis Foundation

Objectives: Multiple lines of evidence, including population-basedstudies, demonstrate that tea consumption, especially green tea, isassociated with reduced risk of cancer and other chronic diseases. Onemechanism for the biological effects of Camellia sinensis is attributableto polyphenols that suppress tumor angiogenesis, the process of bloodvessel formation that drives tumor progression. It is unknown, however,if there are differences in potency among teas. We studied Chinese,Japanese, and English teas for their antiangiogenic activity through theuse of standard in vitro methods in drug discovery and compared theirpotency.

Methods: Loose leaf teas were prepared by conventional hot waterseeping, and were then cooled and filter sterilized. Teas examined wereChineseDragon Pearl Jasmine, ChineseDragonwell Lung Jing, JapaneseSencha, English Earl Grey, and a blend of teas. Human umbilical veinendothelial cells were grown on monolayers in Matrigel, and incubatedwith tea extracts. Image analysis of capillary tubes was performed andcapillary tubes, tube length, tube area, and branch point nodes werequantified. An angiogenesis metric was devised based on IC50 andlevel of suppression of capillary tubes, which allowed comparison ofbioactivity among the teas.

Results: Conventionally, green teas are thought to possess greaterbioactivity than black teas, but we found all tea types tested, includingEarl Grey, possessed a significant amount of antiangiogenic activity.Lung Jing tea had greater bioactivity than Sencha. A custom-blendedtea composition had greater synergistic activity than its individualconstituents.

Conclusions: Tea possesses differential antiangiogenic bioactivitythat has potential to suppress tumor angiogenesis through dietaryintake. Different tea varieties, including both green and black teas,possess a spectrum of bioactivity as quantified by the AngiogenesisPotency Index. This study also revealed the existence of bioactivesynergies in certain combinations of tea varieties.

Funding SourcesSupported byDavid Evans, AdamClayton, Jeffery Tarrant, and Flora

L Thornton Foundation.

Muscadine Grape Extract Inhibits Metastasis Protein RHAMMConcomitant with Reduced Proliferation,Migration, andMolecularSignaling in Triple-Negative Breast Cancer Cells (P08-014)

Marianne Collard, Patricia Gallagher, and E Ann Tallant


Objective: Triple-negative breast cancer (TNBC) is an aggressivebreast cancer subtype with a high propensity to metastasize. Becausethere are no approved treatments to inhibit progression of TNBCmetastasis, we investigated whether a muscadine grape seed and skinextract (MGE) inhibits molecular pathways involved in metastaticTNBC.


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Methods: Proliferation and scratch wound migration of TNBCcells were analyzed with the use of the IncuCyte Zoom system (EssenBioscience). mRNA and protein expression were quantified by reversetranscriptase-polymerase chain reaction andWestern blot, respectively.Cells were treated with 20 μg/mL total phenolics of aqueous MGE.

Results: MGE significantly inhibited proliferation of murine 4T1(68.1%, 24 h; n= 3) and humanMDA-MB-231 (44.3%, 48 h; n= 3) andBT-549 (29.0%, 48 h; n= 3) TNBC cells. MGE alsomarkedly attenuatedmigration of TNBC cells, independent of proliferation, by 59.6% in4T1 cells, 29.3% in MDA-MB-231 cells, and 44.2% in BT-549 cells(12 h, n = 3). The hyaluronan-mediated motility receptor (RHAMM)participates in cell proliferation and migration, is implicated in themetastatic potential of TNBC, and is associated with a poor prognosis.MGE significantly reduced RHAMM protein expression (4T1: 55.5%,12 h, n = 3; MDA-MB-231: 83.7%, 36 h, n = 4; BT-549: 80.7%, 36h, n = 3) and HMMR mRNA, the gene encoding RHAMM (MDA-MB-231: 75.4%, 36 h, n = 3; 4T1: 82.4%, 12 h, n = 3), suggestingtranscriptional regulation. A retinoblastoma protein (RB) and E2Fcomplex regulates HMMR; MGE reduced the inactive phospho-RB inMDA-MB-231 cells (83.9%, 36 h, n = 3). Cyclin D1, a regulator of RBphosphorylation, was also decreased byMGE inMDA-MB-231 and 4T1cells (88.7%, 36 h, n = 3 and 88.8%, 12 h, n = 5, respectively). CyclinD1 expression is controlled by multiple signaling pathways, includingAKT andMAPK. Interestingly,MGEprimarily inhibitedAKT signalingin MDA-MB-231 cells and MAPK signaling in 4T1 cells, suggestingdifferential regulation of molecular pathways that control the cyclinD1/phospho-RB/HMMR pathway in TNBC.

Conclusions: Our findings suggest that MGE inhibits multiplesignaling pathways that converge to reduce RHAMM expression. SinceRHAMM is a crucial protein in TNBC metastasis, our results suggestthat MGE may be a novel medical food for the effective treatment ofTNBC metastasis.


Postprandial Glucose and Insulin Responses to Blueberries(Vaccinium angustifolium) Consumed with a Higher-CarbohydrateBreakfast Meal in Healthy Adults (P08-015)

Adele Corkum,1 Kim Stote,2 Marva Sweeney-Nixon,1 andKatherine Gottschall-Pass1

1University of Prince Edward Island, Canada; and 2State Universityof New York, Empire State College

Objective: Blueberries, as well as the phenolic compounds theycontain, may alter metabolic processes related to glucose regulation.This study investigated the effects of adding 140 g (1 cup) of blueberriesto a higher-carbohydrate breakfast meal on postprandial glucose andinsulin responses.

Methods:Aspart of a randomized crossover design study, 17 healthy(mean ± SD body mass index: 23 ± 3 kg/m2) adults (mean ± SD age:47± 15 y; n= 13 women, n= 4men) consumed a standardized higher-carbohydrate breakfast (290 kcal, 55 g carbohydrate, 6 g fat, 4 g protein)along with 2 treatments: 1) 140 g (1 cup) frozen, then thawed, wholeblueberries; and 2) a placebo gel (matched for calories, sugars, and fiberof thewhole blueberries). Each subject participated in two 2-hmeal tests

on separate visits, >7 d apart. Venous blood samples for glucose andinsulin concentrations were obtained prior to and at 30, 60, 90 and 120min after consuming the breakfast meals.

Results: Plasma glucose concentrations significantly increased frombaseline at 30 min, then declined below the fasting concentration,after the consumption of both blueberry and placebo breakfast meals(P< 0.0001). Serum insulin concentrations significantly increased frombaseline at all time points after the consumption of both blueberryand placebo breakfast meals (P < 0.0001). The glucose and insulinconcentrations peaked at 30 min post breakfast meals and decreasedthereafter. There were no significant differences between the 2 treat-ments with breakfast meals for glucose and insulin concentrations;however, insulin concentrations at 30 and 60min were attenuated in theblueberry treatment group (9% and 16%, respectively). Area under thecurve values for postprandial glucose and insulin concentrations werenot significantly different.

Conclusions: Consumption of blueberries with a higher-carbohydrate breakfast meal did not alter postprandial glucoseresponses, although insulin responses were decreased at 30 and 60 minin healthy adults. Additional research is needed to determine whetherblueberries or other interventions with phenolic compounds improvepostprandial responses in glucose regulation.

Funding SourcesThe Wild Blueberry Association of North America.

Bioavailability and Acute Effects of Bioactive Compounds inWatermelon Juice on Blood Pressure (P08-016)

Kristi M Crowe-White, Tanja Dudenbostel, and Amy Ellis

The University of Alabama

Objective:Hypertension is a modifiable risk factor for cardiovascu-lar morbidity and mortality. Previous investigations with watermelonextracts have shown promise for improving blood pressure following6 wk of supplementation. These beneficial effects were attributed toarginine and citrulline, yet circulating levels of these amino acidsor other bioactive compounds in watermelon were not quantified.Furthermore, the acute effects of watermelon ingestion on bloodpressure have not been evaluated. The objective of this pilot studywas toassess the bioavailability of lycopene, citrulline, and arginine followinga one-time dose of 100% watermelon juice and their subsequent effectson blood pressure in older adult women.

Methods: Women (n = 8) aged 60–70 y were asked to consumea 12-oz serving of 100% watermelon juice after an 8-h overnight fast.At baseline and 2 h after juice consumption, blood was collected andblood pressure measurements were performed according to publishedguidelines. Pulse pressure, a surrogate estimate of arterial stiffness,was calculated as the numeric difference between mean systolic andmean diastolic blood pressure. Serum levels of lycopene, citrulline, andarginine were assessed with the use of a mass spectrometer interfacedwith an ultra-high-performance liquid chromatography system.

Results: Ingestion of one 12-oz dose of 100% watermelon juiceresulted in a 3-fold increase in circulating lycopene levels (P < 0.001);however, no significant differences in amino acids were observedpre- and post-ingestion. Average systolic blood pressure and pulse


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pressure decreased, although these decreases did not achieve statisticalsignificance.

Conclusions: Given the decreases in measures of blood pressure,albeit nonsignificant, data from this pilot study suggest the needfor further investigation of the absorption kinetics of watermelonbioactives in order to establish a dose-duration effect for improvementsin blood pressure.

Funding SourcesThe Academy of Nutrition and Dietetics Healthy Aging DPG.

Oleanolic Acid Induces Autophagy in Neuron Cells throughmTOR Pathways and its Application in a Parkinson Disease AnimalModel (P08–017)

Xiaoli Dong,1 Wen Xuan Yu,1 Xiong Wang,2 Qing Juan Tang,2Qing Zhao,3 and Man Sau Wong1

1The Hong Kong Polytechnic University; 2Ocean University ofChina; and 3 Linzi Maternal & Child Health Hospital of Zibo, China

Objectives: Parkinson disease (PD) is a common neurodegenerativedisease, and autophagy is one of the important PD pathogenic factors atthe molecular level. Oleanolic acid (OA) is a pentacyclic triterpenoidcompound that has been identified in >1620 dietary plants andmedicinal herbs. The present study is designed to delineate themolecular mechanisms by which OA regulated cell autophagy and toinvestigate the potentially neuroprotective actions of OA for treatmentof PD.

Methods: Rat pheochromocytoma PC12 cells were used to in-vestigate the effects of OA on cell autophagy and the under-lying mechanisms. An acute PD animal model was constructedby injecting male C57BL/6J mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 15 mg/kg, intraperitoneal) 4 times in oneday. Oral administration of OA (100 mg · kg–1 · d–1) in the PD micemodel lasted 8 consecutive days.

Results: OA (10 μM) treatment promoted LC3II protein levelsand LC3I to LC3II conversion in rat pheochromocytoma PC12 cells,which are the widely used parameters to characterize autophagosomeformation; OA (10 μM) treatment also significantly increased LC3puncta in PC12 cells, shown by immunofluorescence measurements.An investigation of the mechanism indicated that treatment with3–10 μM OA for 6 h resulted in inhibition of phosphorylation ofmTOR and its downstream 70S6K proteins in PC12 cells, which aspart of the mTOR pathway are important in the regulation of cellautophagy. Furthermore, OA (100 mg · kg–1 · d–1) treatment for 8d did not significantly increase the numbers of tyrosine hydroxylase-immunoreactive neurons in the subtantia nigra pars compacta or thedopamine levels in the striatum in the MPTP-induced acute PD micemodel.

Conclusions: OA can promote the formation of autophagosomesin PC12 cells and positively induce cell autophagy. The potentialmechanism by which OA regulates cell autophagy is related to mTORpathways. OA failed to exert any significant neuroprotective effects inan MPTP-induced acute PD mice model.

Funding SourcesShenzhen Basic Research Program (JCYJ20150630115257900); Na-

tional Natural Science Foundation of China (81601110); National

Natural Science Foundation of China (81528024); Consultancy Grantfrom Linzi Maternal & Child Health of Zibo.

Antioxidant Capacity of Maine Brown Edible Seaweeds andthe Anti-Inflammatory Activity in Lipopolysaccharide-StimulatedRAW264.7 Macrophages (P08-018)

Xue Du and Angela Myracle

University of Maine

Objective: Chronic inflammation is an underlying factor in healthconditions such as diabetes and cardiovascular disease. Seaweeds area group of photosynthetic macroalgae that are known to contain anti-inflammatory bioactive compounds. The objective of this study was toinvestigate the antioxidant capacity and the potential anti-inflammatoryactivity of fresh and commercially available dried seaweed [Ascophyllumnodosume (AS), Laminaria digitata (LD), and Alaria esculenta (AE)]harvested in Maine.

Methods: Commercially dried and freeze-dried seaweeds (fresh)were extracted with 80% methanol. Total phenolics were quantifiedby the Folin-Ciocalteu assay. The antioxidant capacity was evalu-ated with DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Cytotoxicityof seaweed extracts at different levels was examined via MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay. Theanti-inflammatory activity of seaweed was assessed by the inhibitionof nitric oxide production in lipopolysaccharide (LPS)-stimulatedRAW264.7 macrophages.

Results: Compared with commercially dried seaweeds, fresh sea-weeds contained higher amounts of phenolic compounds and exhibitedstronger antioxidant capacity. FreshAShad the highest phenolic content(21.94 mg gallic acid/g seaweed) and strongest antioxidant capacity(16.33 mg gallic acid/g seaweed). The concentrations of extracts at 600µg/mL did not lower the viability of RAW264.7 cells. AS exhibitedthe highest inhibitory activity of nitric oxide production in LPS-stimulated macrophages. At 600 µg/mL, AS extract inhibited nitricoxide production by 39.8%. The inhibitory effects on nitric oxideproduction of LD and AE were minor.

Conclusions: AS exhibited higher antioxidant capacity and betteranti-inflammatory potential comparedwith LD andAE. Fresh seaweedshad higher bioactivity than commercially dried seaweeds.

Fatty Liver–DrivenApoptosis Due toHigh-Fat Diet in an SAMP8Aging Model Is Alleviated by Administration of a Potato BioactivePeptide (P08–019)

Stanley DUmeUS,1 Wei-Wen Kuo,2 and Chih-Yang Huang2

1State University of Haiti Faculty of Medicine and Pharmacy; and2China Medical University

Objective: Nonalcoholic fatty liver disease (NAFLD) has become agrowing epidemic inWesternized-lifestyle population groups. Previousstudies revealed a potential lipolysis-stimulating role for oral supple-mentation of a potato-extracted crude-protein hydrolysate (CPP) in ayoung adult murine model. Given that peptide segments exhibit highlyefficacious advantages due to high absorption and low power, comparedto parent protein, we hypothesized that intraperitoneal delivery of


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a purified bioactive peptide from CPP to high-fat diet (HFD)-fedSAMP8 aging mice would effectively modulate hepatic targets involvedin lipolysis-promoting activities, and hence confer protection againsthepatocyte apoptosis triggered by lipotoxicity.

Methods:The peptideDIKTNKPVIFwas purified, synthesized, andused alongside its parent protein (CPP) and the hypolipidemic drugprobucol. Six-month-oldmicewere divided into 5 groups (n= 4/group)for a 14-wk experimental design. The normal control group (CHO)was fed a standard chow until they were killed. After 6 wk of HFD(60% calories from fat), the remainingmice groupswere treatedwithout(HFco) or simultaneously with either DIKTNKPVIF peptide (HF-DF;15 mg/kg body wt, q24h × 8 wk, intraperitoneal), CPP (HF-CPP; 50mg/kg body wt, q24h × 8 wk, via oral gavage) or probucol (HF-Pr; 500mg/kg body wt, q24h × 8 wk, via oral tube) for the final 8 wk.

Results: Compared with CHO, histopathologic analyses of livertissue samples revealed massive hepatic fat accumulation in HFco, butreduced fatty changes in HF-DF. In contrast to HFco, the numbersof terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive hepatic cells were considerably lower in HF-DF,indicating: 1) reduced macro- and microvesicular fatty changes; and, atthe protein level, 2) upregulation of the SIRT1/PGC-1α axis (P< 0001)and survival markers such as p-Akt (P < 0,01) with concomitantinhibition of proapoptotic activity promoted by JNK signaling.

Conclusions: The present data indicate that intraperitoneal admin-istration of our synthesized peptide in aging mice effectively attenuatedHFD-induced steatosis and hepatocyte apoptosis through enhancingthe SIRT1/PGC-1α axis. Therefore, DIKTNKPVIF peptide may beproposed as potential therapeutic strategy capable of ameliorating thehepatocellular injuries that underlie NAFLD in elderly.

Funding SourcesThis study was financially supported in part by grants from the

Ministry of Science and Technology of Taiwan (MOST103-2410-H-029-037 and MOST104-2410-H-029-033-MY2). We express ourgratitude to the TaiwanMinistry of ForeignAffairs (MOFA) ScholarshipProgram for the Republic of Haiti, through which SD (Faculte deMedecine et de Pharmacie, Universite d’Etat d’Haiti—FMP UEH) wassupported.

Emodin Inhibits Pathologic Cardiac Hypertrophy via Inhibitionof Histone Deacetylase Activity (P08-020)

Levi W Evans, Leah Burnett, Bradley Ferguson

University of Nevada, Reno

Objectives: Diet is a central pathogenetic element in the develop-ment of heart disease and heart failure. Indeed, studies have linked dietto regulation of genes that promote pathologic cardiac enlargement.Emerging data suggest that this diet-gene interaction is mediated byepigenetic regulation. The aim of this study was to test the hypothesisthat dietary histone deacetylase (HDAC) inhibitors stop pathologiccardiac hypertrophy via epigenetic regulation of gene expression.

Methods: To test our postulate, we used neonatal rat ventricularmyocytes (NRVMs) stimulated with phenylephrine (PE, 10 μM) orphorbol myristate acetate (PMA, 50 nM) to induce receptor- orintracellular-mediated pathologic cardiac hypertrophy. NRVMs werefurther cotreated with vehicle or one of 18 dietary HDAC inhibitors

(10 μM) previously identified by our laboratory. The classic pan-HDAC inhibitor Trichostatin A (TSA, 200 nM) was used as a positivecontrol. NRVMs were treated for 48 h. Cells were then fixed forimmunostaining of cell size, lysed for protein expression examiningHDAC activity and histone acetylation, or isolated for RNA sequencingand quantitative PCR. A minimum of 3 experiments, with an n ≥ 3per group was performed and data were quantified and normalized perstandard procedures. One-way ANOVA with Tukey’s post-hoc test wasperformed. P ≤ 0.05 was considered significant.

Results: Of the 18 dietary compounds, 6 attenuated pathologiccardiac hypertrophy in response to PE and PMA. However, onlyemodin significantly inhibited HDAC activity that corresponded toincreased histone acetylation in NRVMs. Similar to the classic pan-HDAC inhibitor TSA, emodin attenuated hypertrophic gene expres-sion, suggesting an epigenetic role for this bioactive compound. It wasnoteworthy that rhubarb, which is enriched in emodin, was also foundto inhibit cardiac hypertrophy and HDAC activity in NRVMs.

Conclusions: Five-year mortality rates following heart failure diag-nosis are ∼50%, highlighting a critical need for improved therapeuticstrategies. Findings from our study highlight emodin and emodin-enriched foods as cardioprotective via epigenetic regulation of globalgene expression. Thus, these data point to epigenetic modifiers in ourdiet that have the potential to prevent or treat heart disease.

Funding SourcesThis work is supported by the USDA National Institute of Food

and Agriculture (Hatch-NEV00727) and the Dennis Meiss & JanetRalston Fund for Nutri-epigenetic Research to BSF. Core facilitiesused for Research were supported by National Institute of GeneralMedical Sciences of the National Institutes of Health under grant P20GM103554.

Thermogenic Activity of Microbial Metabolites Produced byLactobacillus plantarumWCFS1 from Tannins in Gnotobiotic Mice(P08-021)

Chuo Fang, Hyemee Kim, Lora Yanagisawa, Maritza Sirven,Robert Alaniz, Stephen Talcott, andSusanne Talcott

Texas A&M University

Objectives: Intestinalmicrobialmetabolites from tannins, includinggallic acid (GA) and pyrogallol (PG), may possess potential antiobesityproperties that lower the risk of metabolic disorders. Lactobacillusplantarum, found in the intestinal microbiome, possess enzymaticactivities that degrade tannins into GA and PG. In this study, theanti-inflammatory, antidiabetic, and thermogenic activities of orallyadministered tannins in the presence or absence of L. plantarum werecompared with PG in plasma and adipose tissues in gnotobiotic micefed a high-fat diet (HFD).

Methods: Germ-free (GF) C57BL/6J mice were randomly dividedinto 4 groups; 1 group was colonized with L. plantarum WCFS1 andreceived tannins. Noncolonized groups received a control treatment,PG and tannins, respectively for 6 wk. All mice were fed an HFD fromweek 2 to week 6. After 6 wk, plasma, epididymal white adipose tissue(eWAT) and interscapular brown adipose tissue (iBAT) were collected.Biomarkers for inflammation, insulin resistance, and thermogenesiswere assessed in plasma, eWAT, and iBAT.


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Results: Colonization of L. plantarum in tannin-fed mice signifi-cantly reduced plasma levels of insulin, gastric inhibitory polypeptide(GIP), glucagon and pancreatic polypeptide (PP) by 60%, 40%, 50%,and 50%, respectively, whereas PG reduced GIP and glucagon by35% and 47%, respectively, indicating reduced insulin resistance. IneWAT and iBAT, tannins but not PG, significantly increased theexpressions of thermogenic genes (PGC-1α, Cox2, PRDM16, Cox7a1,Cidea, Tmem26, Tbx1, and CD137) and AMPKα1; colonization of L.plantarum induced a further increase in thermogenesis and AMPKactivity.

Conclusions: These data indicate that L. plantarum reducesinsulin resistance and promotes thermogenesis potentially throughtannin-metabolizing enzymatic activities yielding absorbable bioactivemetabolites includingGAandPG. This study suggests a potential role ofsubstrate-probiotic interactions in microbial tannin metabolism in theprevention of diet-induced insulin resistance, and further investigationof their roles in thermogenesis is needed.

Funding SourcesThis study was supported by grants from the National Mango Board

(Orlando, FL).

Fat-Lowering Effects of Green Coffee Bean Extract and 3-O-Caffeoylquinic Acid Are Dependent on SBP-1 and DAF-16 inCaenorhabditis elegans (P08-022)

Renalison Farias-Pereira and Yeonhwa Park

University of Massachusetts Amherst

Green coffee bean extract (GCBE) is used for weight managementand reports suggest that its effect on weight control is associated withits major phenolic compounds, chlorogenic acids. However, there aredifferent reports of the relative efficacy of GCBE and chlorogenic acidson fat accumulation. Since GCBE comprises ∼50% chlorogenic acids,among which 3-O-caffeoylquinic acid (3-CQA) is one main form, weinvestigated the mechanism of actions of GCBE and 3-CQA on fataccumulation with the use of Caenorhabditis elegans. Treatment withGCBE (5 mg/mL) and 3-CQA (2.65 mg/mL) significantly reduced fataccumulation by 29% and 23%, respectively, compared with the control.The effects of GCBE and 3-CQA on fat accumulation were abolishedin mutants of sbp-1 (an ortholog of mammalian sterol-regulatory-element-binding protein) and daf-16 (an ortholog of mammalianForkhead box O transcription factor), which suggests that both sbp-1 and daf-16 are important genes in the effects of GCBE and 3-CQAon fat metabolism. We further confirmed that GCBE and 3-CQAinduced nuclear translocation of DAF-16 and significantly reduced theexpression of sbp-1 and pod-2 (a homolog of acetyl-CoA carboxylase),a downstream target of sbp-1. Therefore, these results suggest thatGCBE and 3-CQA reduce fat accumulation via SBP-1- and DAF-16-dependent mechanisms in C. elegans, which are involved in lipogenesisand the insulin-signaling pathway, respectively. These results suggestthat chlorogenic acids are most likely responsible for GCBE’s effects onfat metabolism.

Funding SourcesThis work was financially supported by CNPq, Conselho Nacional

de Desenvolvimento Científico e Tecnológico—Brasil (National Coun-sel of Technological and Scientific Development—Brazil).

Blueberry Polyphenols Decrease Oxidative Stress and Increasethe Levels of Nitric Oxide Metabolites in Angiotensin II–StimulatedHuman Aortic Endothelial Cells (P08-023)

Rafaela G Feresin,1 Hannah Huff,2 Rami Najjar,1 Shengyu Mu,2and Joshua Phelps2

1Georgia State University; and 2University of Arkansas for MedicalSciences

Objective: The aim of this study was to investigate the mechanismsby which blueberry (BB) polyphenols improve vascular endothelialfunction in angiotensin II (Ang II)–stimulated human aortic endothe-lial cells (HAECs).

Methods: BB polyphenol extract (BBPE) was prepared throughthe use of 70% methanol extraction followed by purification withchloroform and separation with ethyl acetate. HAECs were treated withand without 200 µg/mL of BBPE for 12 h prior to stimulation with AngII for 8 and 12 h. Nitric oxide (NO) metabolites were determined witha colorimetric kit. Levels of reactive oxygen species (ROS) were deter-mined after 30-min incubation with 2′,7′-dichlorodihydrofluoresceindiacetate. Expression of phosphoendothelial NO synthase (eNOS),catalase (CAT), glutathione peroxidase (GPx1) as well as superoxidedismutase (SOD) 1 and SOD2 were assessed by western blot. Resultswere analyzed by ANOVA followed by Tukey-Kramer post-hoc test.

Results:BBPE increasedNOmetabolite levels (1.3± 0.14 comparedwith 0.66 ± 0.11, P = 0.01) and expression of phospho-eNOS(3.58± 0.89 compared with 1.28± 0.09, P= 0.04) in Ang II–stimulatedcells compared with Ang II alone. ROS levels were decreased in AngII–stimulated cells pretreated with BBPE compared with Ang II alone(0.70 ± 0.06 compared with 1.07 ± 0.05, P = 0.03). GPx1 expressionwas increased by BBPE in the presence of Ang II compared with AngII alone (3.67 ± 0.74 compared with 1.14 ± 0.23, P = 0.05). SOD2expression was increased in Ang II–stimulated cells compared withcontrol (3.82 ± 0.96 compared with 1.00 ± 0.00, P = 0.04), no furtherchanges in expression of SOD2 were observed following the addition ofBBPE. Lastly, no significant changes were observed in SOD1 and CAT.

Conclusion:Altogether, our data partially elucidate themechanismsby which BB polyphenols exert their protective effects on the endothe-lium as we demonstrate that BBPE increases not only the productionof NO but also its bioavailability as it decreases ROS by increasing theexpression of antioxidant enzymes.

Funding SourcesUAMS-CHP-Dean’s Society Grant.

Novel Compartmental Analysis to Estimate β-Hydroxy-β-Methylbutyrate Production and Conversion Rates in Humans(P08-024)

Parisa Ghane, Marielle Engelen, Gabriella AM Ten Have, JohnJ Thaden, Ulisses M Braga-Neto, Ivan Ivanov, and Nicolaas EPDeutz


Objective: β-Hydroxy-β-methylbutyrate (HMB) is a metabolite ofα-ketoisocaproate (α-KIC), which is produced by transamination ofleucine. Restoring reduced plasma HMB concentrations in older adultshas attracted interest as a means to reduce sarcopenia. To obtain insight


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into the metabolic pathways of HMB, we developed a new approach toreplicate the metabolism of leucine, α-KIC, and HMB in humans thatuses a 6-compartment model.

Methods:After intravenous administration of a 8.5-mL pulse of thestable isotopes of leucine, α-KIC, and HMB, blood was sampled for 4 hin 20 older adults. The plasma enrichment data were subsequently fittedwith the 6-compartmentmodel. Themodelwas developed, sequentially,in 3 steps: 1) describing the kinetics of each substrate by a distinct 2-compartment model; 2) simulating the products of metabolites relativeto each substrate through the use of the convolution integral for lineartime-invariant systems; and 3) computing intracellular pool size (Q2),rate of appearances (Ra), and de novo productions from compartmentalmodels and interconversion fluxes from area under the curves (AUCs).The highlights of our method include its efficient running time,robustness, reduction in regression parameters, and capability to modelany metabolic pathways. We used the R packages quadprog to calculateinitial values and minpack.lm for the Levenberg-Marquardt algorithmincorporatedwithin the iterative nonlinear regression. All quantities arein mean ± SE.

Results: Goodness of parameters was assessed by model coefficientof variation (m-CV). Mean ± SE percentage of m-CV over thepopulation for leucine, α-KIC, and HMB were 11.14 ± 3.14%, 22.97± 4.42%, and 9.76 ± 1.47%, respectively. Q2 and Ra are estimated inµmol/kg body wt and µmol · kg body wt–1 · min–1: Q2 = 373.657± 66.084 µmol/kg body wt and Ra = 2.695 ± 0.074 µmol · kg bodywt–1 · min–1 for leucine; Q2 = 16.133 ± 3.939 µmol/kg body wt andRa = 0.130 ± 0.014 µmol · kg body wt–1 · min–1 for KIC; Q2 = 0.4136± 0.0468 µmol/kg body wt and Ra= 2.553× 10–3 ± 3.34× 10–4 µmol ·kg body wt–1 · min–1 for HMB. Intracellular conversion of leucine to α-KIC is 112.318± 18.663, α-KIC to leucine is 18.208± 9.258, and α-KICto HMB is 0.056 ± 0.010.

Conclusions: We developed a compartmental model to measureconversion rates of leucine into α-KIC, and α-KIC into HMB. The lowintracellular conversion of leucine to HMB of 0.1% needs to be takeninto account when considering dietary leucine to restore HMB plasmaconcentration in older adults.

Funding SourcesNone.

Compartmental Data Analysis to DetermineWhole-Body Short-Chain Fatty Acid Production through the Use of a Pulse of StableTracers in Humans (P08-025)

Parisa Ghane, Sarah Kirschner, Marielle PKJ Engelen, IvanIvanov, Ulisses M Braga-Neto, and Nicolaas Deutz


Objective: Short-chain fatty acids (SCFAs) are important to main-tain intestinal health. In this study, we estimated the SCFA productionof the human microbiome via a pulse of stable tracers and through theuse of compartmental data analysis.

Methods: A 2-compartment model, containing plasma and tissuepools, was designed to describe extra- and intracellular kinetics ofSCFA acetate (C2), propionate (C3), and butyrate (C4). We gave 14

healthy older adults an 8-mL pulse containing [13C2]-acetate, [1–13C]-propionate, and [1–13C]-butyrate, and sampled plasma regularly for1 h. We measured the tracer enrichments in plasma by GC-MS/MS.The open-source software R was used to estimate SCFA de novointracellular production (F02) and rate of appearance (Ra) through 3major steps: 1) fitting the function f(t) = Aexp(–at) + Bexp(–bt) onexperimental tracer-tracee ratios; 2) development of the tracer model;and 3) calculations of the tracee parameters. We used the curve-peelingapproach, the Levenberg-Marquardt algorithm, outlier detection, andtuning up of convergence criteria which significantly reduced runningtime and optimized the functions’ parameters. After an evaluationof goodness of fit, a-priori identifiable models for tracer and thentracee were developed based on physiologic considerations and thefitted f(t).

Results: The function f(t) was fitted independently for each subject.Precision of the functions’ parameters was assessed bymodel coefficientof variation. The high precision obtained (Table 1) for the parametersillustrates the goodness of fit. Tracee parameters [F12 flux fromextracellular (Q1) to intracellular compartment (Q2)], show an 8- to 12-fold higher intracellular production (F02) of the SCFA than estimatedfrom the rate of appearance (Ra), which is usually measured with aprimed constant infusion protocol (Table 2).

Conclusions: We developed a compartmental analysis approachto estimate SCFA transport between extracellular and intracellularcompartments and intracellular production in humans.We hypothesizethat compartmental analysis estimates the SCFA production in themicrobiome, mainly present in the large intestine.

Funding SourcesNone.

TABLE P08-025-1

TABLE P08-025-2


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Dietary Malate or Lactate Attenuates High-Fat Refined Diet–Induced Adiposity, Insulin Resistance, and Fatty Liver in MaleC57BL/6J Mice (P08-026)

John Gieng,1 Shiva Barforoshi,2 Hellen Ouyang,2 and Mark KShigenaga2

1San José State University, CA; and 2Children’s Hospital OaklandResearch Institute, CA

Objective: Organic acids, such as malate and lactate, may beimportant contributors to health because they are known to promoteenergy metabolism. Examples of foods that contain abundant amountsof these compounds include fruit and fermented food products. Thesedietary “energy acids” may confer some of the health benefits normallyassociated with these foods (e.g., normalizing insulin sensitivity, weightmaintenance, and metabolic health). The objective of this study wasto determine, in a diet-induced obesity mouse model, the metaboliceffects of two energy acids (malate and lactate) when provided throughdrinking water.

Methods: Male C57BL/6J mice (n = 36, 9/group) consumed adlibitum for 16 wk a standard rodent diet or a high-fat refined diet(HFRD, 45.9% kcal fat) with drinking water alone, or a HFRD withdrinking water supplemented with either malate or lactate at a food-relevant concentration (100 mM, titrated to pH 7.4 with potassiumhydroxide). At the end of 16 wk, body weight, adiposity, insulinresistance, dyslipidemia, and hepatic lipid content were measured.

Results: Body weight (P < 0.05) and adiposity (visceral, perirenal,epididymal, and subcutaneous, P < 0.05) increased with HFRDfeeding compared with the standard diet. They were also moreinsulin resistant (decreased homeostasis model assessment of insulinresistance, P< 0.01) and had higher plasma total cholesterol (P< 0.01)but not plasma triglycerides (P > 0.05), although hepatic triglycerideswere higher (P < 0.01). Both malate- and lactate-supplemented miceon the HFRD had blunted weight gain (P < 0.05) and adiposity(P < 0.05). Moreover, mice supplemented with either energy acid weremore insulin sensitive (P< 0.05) and had lower plasma total cholesterol(P < 0.05) compared with HFRD feeding alone. Finally, the rise inhepatic triglycerides with HFRD feeding was attenuated when micewere supplemented with either malate (P < 0.05) or lactate (P < 0.05).

Conclusions: Our data suggest that supplementation with food-relevant levels of either malate or lactate protects mice from HFRD-induced weight gain, insulin resistance, and dyslipidemia. This impliesthat the metabolic benefits attributed to health-promoting foods suchas fruit or fermented products may in part be due to their energy acidcontent and should therefore be considered when assessing a food’snutritional quality.

Funding SourcesBruce and Giovanna Ames Foundation.

Flavanols from Grape Seed and Pine Bark Protect againstObesity-Induced Adipose Inflammation (P08-027)

Laura E Griffin, Dane Fausnacht, Jessica L Tuzo, Adele Adding-ton, Katherine Racine, Haiyan Zhang, Michael Hughes, SeanO’Keefe, Andrew Neilson, and Amanda Stewart

Virginia Tech

Objectives: Flavanols are present in foods at a wide range of degreesof polymerization (DP) of the catechin monomers. Weight gain andobesity come with an increased risk for several chronic diseases, andflavanol consumption may ameliorate both obesity and the resultingeffects such as inflammation. This study was designed to examinewhether flavanols with differentmean degrees of polymerization (mDP)have distinct impacts on markers of high-fat (HF) diet–inducedmetabolic syndrome when they are consumed at low doses.

Methods: Grape seed extract (GSE) and pine bark extract (PBE)with mDPs of 2.1 and 2.7 were employed. C57Bl/6J mice were givenan HF diet supplemented with 35 mg · kg body wt–1 · d–1 GSE orPBE based on crude extract weight or flavanol concentration for 13wk. Mice on low-fat (LF) diet and HF diet without extracts served ascontrols.

Results: All flavanol groups and the HF control incurred signifi-cantly higherweight gain than the LF control. Flavanol supplementationdid not prevent weight or fat gain relative to HF control. Improvementsto fasting blood glucose levels were observed only in the PBE groupdosed on flavanol concentration after 10 wk but in both PBE groupsafter 11 wk. All treatment groups exhibited significantly lower adiposelevels of interleukin (IL)-6 compared with HF control, regardless ofmDP and despite the significantly higher weight gain compared withcontrols. Most notable was the GSE-supplemented group, which hadthe lowest IL-6 levels but the highest overall weight gain. Similar trendswere observed in tumor necrosis factor α levels, but the PBE groupsdemonstrated a greater cytokine-lowering effect. These findings suggestthat flavanols may prevent against obesity-associated inflammationwithout concurrent physiologic improvements.

Conclusions: Flavanol supplementation was not able to preventweight gain regardless of mDP. Despite the lack of physiologic improve-ments, flavanols were able to reduce inflammatory cytokine production,which is typically associatedwith othermetabolic derangements. Largerflavanols may be more effective as they demonstrated the ability toreduce inflammatory cytokines and prevent derangement to fastingblood glucose.

Funding SourcesFunding for this work was provided, in part, by the Virginia

Bioinformatics Institute and Fralin Life Science Institute ExploratoryGrant and by the Virginia Agricultural Experiment Station and theHatch Program of the National Institute of Food and Agriculture,USDA.

A Comparative Study of the Anti-Inflammatory Effects of BerryPhenolics and Volatiles (P08-028)

Inah Gu, Cindi Brownmiller, Luke Howard, and Sun-Ok Lee

University of Arkansas

Objectives: The aim of this study was to investigate the anti-inflammatory effect of berry volatiles and phenolics through the useof a lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cellmodel by measuring the modulation of production of nitric oxide(NO), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α).We hypothesized that berry volatiles have anti-inflammatory effectscomparable to those of berry phenolics, modulating cytokines andproinflammatory factors.


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Methods: RAW264.7 cells were pretreated with 3 different dilutions(diluted 50-, 100-, and 200-fold) of 6 berry (blackberry, black raspberry,blueberry, cranberry, red raspberry, and strawberry) phenolic or volatilefractions for 1 h. Cells were then incubated with or without LPS (100ng/mL) for 24 h. The NO production was measured through the use ofthe Griess reaction. The levels of IL-6 and TNF-α in the culture mediawere quantified by ELISA kits. All data analysis was performed withthe use of SAS version 9.4 to determine the statistical significance, withP < 0.05 considered significant. Data were presented as mean ± SEM.

Results: Results showed that berry phenolics and volatiles signifi-cantly decreased the LPS-induced NO production by 38–93% and 47–79%, respectively (P < 0.01). There were no significant differencesamong 3 different dilutions of berry phenolics and volatiles in theRAW264.7 cells. Except black raspberry, berry volatiles decreased thelevel of TNF-α and IL-6 in LPS-stimulated RAW264.7 cells, comparableto berry phenolics.

Conclusions: Results from this study suggest that volatiles from 6common berries have anti-inflammatory activities comparable to thoseof phenolics in vitro.

Funding SourcesArkansas Bioscience Institute.

Bioequivalence Study on Vitamin C Gummy and Caplet inHealthy Adults (P08–029)

Najla Guthrie,1 Malkanthi Evans,1 Janaki Iyer,1 MohammedMarzuk,1 Kelly H Zhang,2 William Hooper,2 and AnnahitaGhassemi2

1KGK Science Inc.; and 2Church & Dwight

Objective: The aim of this study was to evaluate the bioequivalenceof vitamin C gummy and a comparator vitamin C caplet in healthyadults.

Methods:The designwas a randomized examiner-blind comparatorcontrolled crossover studywith 2 sequences separated by a 7-dwashout.Healthymale or female adults 18–55 y of age, bodymass index 18.5–29.5kg/m2 were enrolled. Intake of citrus foods and foods and beveragesfortified with vitamin C was restricted 7 d prior to each dosing.Participants were randomly allocated to 2 interventions: VitafusionPower C gummy (1000 mg) or Nature Made Vitamin C caplet (1000mg). Blood samples were collected prior to dosing and at 0.5, 1, 2, 3,4, 5, 6, 8, 10, 12, and 24 h post-dose for plasma and white blood cell(WBC) and urine was collected prior to dosing and between 0–2, 2–4, 4–8, 8–12 and 12–24 h post-dose for l-ascorbic acid analysis. Safetymeasurements included vital signs, hematology, and clinical chemistry.Subjects provided 7-d food records to ensure compliance to a low-vitamin C meal plan. Standardized meals were provided on in-clinicdays. Statistical methods included a repeated-measures ANOVA.

Results: A total of 30 subjects (9 males, 21 females) mean age34.0 ± 11.4 y and mean BMI 24.5 ± 3.6 kg/m2 completed the study.There were no clinically relevant adverse events and safety parametersremained within the normal clinical range for both products. Bothgummy and caplet demonstrated similar plasma absorption profiles.There were no statistically significant differences between gummy andcaplet in plasma l-ascorbic acid total area under the curve (AUC0–24),and Tmax. Although the caplet elicited a significantly higher Cmax than

the gummy (P < 0.05), the difference in Cmax for gummy and capletwas numerically small. WBC l-ascorbic acid total AUC0–24 and Cmax

were not significantly different between gummy and caplet. Urine levelsof l-ascorbic acid were not significantly different between gummy andcaplet at any measured time point.

Conclusions: Under the conditions of this study, both gummy andcaplet exhibited similar bioavailability and were therefore considered asbioequivalent. This study supports the gummy as a viable alternative tothe caplet for providing vitamin C nutrient.

Funding SourcesChurch & Dwight Co., Inc., Ewing, NJ.

Effects of Mixed-Nut Supplementation on Inflammation, Oxida-tive Stress, Antioxidant Enzymes, and Liver Function Markers inRats Fed an Atherogenic Diet (P08-030)

Mee Young Hong, Amanda Marx, and Caitlin Rasmussen

San Diego State University, CA

Objectives: Cardiovascular disease (CVD) is the number one causeof death in the United States. Inflammation and oxidative stressare key processes in the development of CVD. Nuts are rich inunsaturated fatty acids, plant protein, fiber, vitamins, minerals, andmany phytochemicals. These bioactive materials and nutrients improveantioxidant capacity andhave anti-inflammatory abilities, whichmay beassociatedwith the beneficial effects of nuts onCVD risk factors. Severalstudies have examined the effects of individual nuts on inflammation,but there is limited research on whether the beneficial effects wouldbe extended with the consumption of a mixture of nuts, which wouldprovide a greater nutrient variety. Therefore, the objective of this studywas to examine the effects of mixed-nut consumption on inflammation,oxidative stress, antioxidant capacity, and liver function enzymes in ratsfed an atherogenic (high in fat, cholesterol, and sugar) diet.

Methods: Male Sprague-Dawley rats aged 28 d (total n = 30) wereallocated into 3 groups and fed isocaloric control diet (no nuts), 8.1%pistachio diet (single nut), or 7.5% mixed-nut diet (almonds, cashews,pecans, pistachios, walnuts, peanuts, macadamia nuts, and brazil nuts)for 8 wk.

Results: Both pistachio and mixed-nut groups significantly de-creased C-reactive protein as an inflammation marker (P = 0.045) andthiobarbituric acid reactive substances as an oxidative stress indicator(P = 0.004), and increased total antioxidant capacity (P = 0.048)compared with the control group. Mixed-nut groups had greatersuperoxide dismutase (P = 0.004) and catalase (P = 0.044) and loweraspartate aminotransferase (P= 0.048) activities than the control group.There were no significant differences on alanine aminotransferase,alkaline phosphatase, lactate dehydrogenase, and creatinine kinaseamong groups.

Conclusions: This study suggests that mixed-nut consumptionprovides health benefits comparable to those found for individual nutintake with regard to risk factors for CVD, including inflammation, ox-idative stress, total antioxidant capacity, antioxidant enzyme activities,and liver function markers in atherogenic-diet fed rats.

Funding SourcesAmerican Heart Association (16GRNT31360007).


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Effects of Storage Condition on Bioactive Compound ContentsandAntioxidantActivity of BlackChokeberry (Aroniamelanocarpa)(P08-031)

Eun-sun Hwang and Nhuan Do Thi

Hankyong National University, South Korea

Black chokeberry, Aronia melanocarpa, is termed a “super fruit,”because it is a good source of bioactive phytochemicals and offersvarious health benefits. It has been reported to have anticancer andantimutagenic activities and blood pressure–lowering properties. Theeffect of storage on the content and antioxidant activity of this fruit’sbioactive compounds were evaluated by storing black chokeberry forup to 20 wk (0, 4, 8, 12, 16, and 20 wk) at three different temperature:refrigerated (4°C), freezing (–20°C) and deep-freezing (−75°C). Thetotal polyphenol contents of black chokeberry extract stored at 4°Cwere decreased from 905.4 to 658.0 μg gallic acid equivalent/g dryweight after 20 wk storage. At −75°C, the total polyphenol contentsof black chokeberry extract were decreased less than at 4°C and−20°C. The total flavonoid contents of black chokeberry extractstored at −75°C were reduced from 423.5 to 351.3 μg quercetinequivalent/g dry weight after 20 wk. Four anthocyanin peaks weredetected in black chokeberry representing, cyanidin-3-O-galactoside,cyanidin-3-O-glucoside, cyanidin-3-O-arabinose, and cyanidin-3-O-xylose based on HPLC analysis. The extractable anthocyanins as well aspolyphenol contents in black chokeberry were decreased by increasingstorage period at both 4°C and −20°C. Longer storage periods atambient temperature may reduce the levels of bioactive compounds.Storage temperatures also had an effect: the highest amounts of totalpolyphenols, flavonoids, and anthocyanins were detected in blackchokeberry stored at 4°C, followed by the −20°C and −75°C samples.We tested the DPPH (2,2-diphenyl-1-picrylhydrazyl), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical, and superoxideanion–scavenging activities, as well as the reducing power activity ofblack chokeberry extract. Samples frozen at −75°C showed strongerantioxidant activities than the other storage temperatures. Based onthese results, freezing at −75°C can help preserve the nutritive value ofblack chokeberry by maintaining high levels of anthocyanins and otherbioactive compounds.

Funding SourcesThis research was supported by Basic Science Research Pro-

gram through the National Research Foundation of Korea (NRF-2016R1A2B4014977).

Phytochemical Analysis, Nutrient and Vitamin Composition ofThaumatococcus danielli Leaves (P08-032)

Adaku Vivien Iwueke,1 Kelechi Osuocha,1 Nnamdi JEjekwumadu,2 and Callista Akalonu1

1Imo State Polytechnic, Umuagwo, Nigeria; and 2Kampala Interna-tional University, Uganda

Objective: Thaumatococus daniellii leaf has for centuries been animportant economic leafwith varied domestic uses. It is principally usedin parts of southern Nigeria for wrapping local foods and delicaciesprior to cooking and fermenting, and also has some folk medicinalattributes, though these lack appropriate scientific validation. The aim

of this studywas to evaluate the bioactive constituents, and to determinethe vitamin and proximate composition of T. daniellii leaves.

Methods: The phytochemical compounds were analyzed by gaschromatography coupled with mass spectrometry (GC-MS), and theproximate and vitamin composition was determined through the useof standard analytic methods.

Results: The GC-MS phytochemical analysis revealed thepresence of 8 bioactive compounds: 1,2,3,4-butanetetrol, d-glycero-d-idoheptose, cyclopentane,1-ethyl-1-methyl-, 6-octen-1-ol,3,7-dimethyl-, 2-octenal, (E)-, octane,1-chloro-,2-acetoxy-1,1,10-trimethyl-6,9-epidioxydecalin, and bicyclo[3.1.1]heptan-3-ol, 2,6,6-trimethyl-, (1α,2β ,3α,5α)-. Vitamin assay results showed that T.daniellii leaf contained 3.1 mg/100 g of vitamin A, 1.07 mg/100 g ofvitamin B-1, 1.32 mg/100 g of vitamin B-3, 1.11 mg/100 g of vitaminB-5, 16.34 mg/100 g of vitamin B-6, 11.86 mg/100 g of vitamin B-12,and 25.19 mg/100 g of vitamin C. The result of the proximate analysisindicated that T. daniellii leaf contains 10.15% ash, 9.67% moisture,20.41% protein, 11.42% lipids, 13.78% fiber, and 34.57% carbohydrate.

Conclusions: Our results suggest that T. daniellii leaves may playcritical medicinal roles in disease management as well as nutritionalroles, and are probably a safer alternative to polyethylene paper wrapswhich have known side effects. Hence the local usage of these leaves asfood wrappers is justified.

Funding SourcesPrivately funded by the authors.

Effects of Fenugreek Seeds on Oxidative Stress, ProinflammatoryCytokines andNitrite as Adjuncts toDiet in Patients withHyperlipi-demia (P08-033)

Poonam Jaglan

Park Hospital, Panipat, India

Background: Fenugreek seeds are rich sources of fiber andpolyphenol antioxidants which are known for hypolipidemic and hy-poglycemic effects and may inhibit atherothrombosis due to their anti-inflammatory, antioxidant, and nitric oxide (NO)–activating effects.

Objectives: In the present trial, we examined the effects of fenugreekseeds on atherosclerotic risk factors, namely inflammation and nitritedeficiency.

Methods: This study was based on a randomized, double-blind,placebo-controlled trial in a hospital. All subjects (n = 61) with hy-perlipidemia were assigned to National Cholesterol Education Program(NCEP) step 1 diet for a period of 12 wk and then randomly assignedto take 2 different test agents in identical sachets for another 12 wk.The test agents were fenugreek seed powder, rich in polyphenols (60.0g/d), and cellulose placebo (3.0 g/d). Of the 61 subjects (total cholesterolbetween 5.17 and 7.76 nmol/L), 60 successfully completed 12 wk of theNCEP step I diet, and then augmented the diet with one of the fiber orfenugreek supplement for an additional 12 wk.

Results: Parameters of oxidative stress, namely malondialdehyde,thiobarbituric acid reactive substances, and diene conjugates, aswell as proinflammatory cytokines, namely tumor necrosis factor α

(TNF-α) and interleukin (IL)-6, showed significant decline from thebaseline levels in the fenugreek group, whereas these changes were


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nonsignificant in the placebo group. The incremental differences fromplacebo for TNF-α (22.5%) and IL-6 (27.8%) were highly significant (P

Conclusions: Treatment with fenugreek seeds revealed significantanti-inflammatory and nitric oxide–activating effects, on NCEP step 1diet effects.

Anti-Inflammatory Effects of Tart Cherry Anthocyanins inZucker Fatty Rats and Cultured Adipocytes (P08-034)

Shasika H Jayarathne,1 April Stull,2 Alexandra Miranda,1 KateClaycombe-Larson,3 Shane Scoggin,1 Iurii Koboziev,1 andNaimaMoustaid-Moussa1

1Texas Tech University; 2University of Maryland Eastern Shore;3USDA-ARS-PA, Grand Forks Human Nutrition Research Center

Objective:Dietary bioactive compounds with antioxidant and anti-inflammatory properties may prevent obesity-associated inflammationand metabolic disorders. Our objective is to understand the mecha-nisms mediating beneficial effects of tart cherry anthocyanins (TCAs)in metabolic disorders. We hypothesized that TCAs reduce obesity-associated inflammation through direct anti-inflammatory effects inadipose tissue.

Methods: Male Zucker fatty rats were fed a diet supplementedwith 4% wt/wt TC powder; the control group fed normal chow dietfor 8 wk. Serum and adipose tissues were collected to determinethe anti-inflammatory effects of TC supplementation. Optimal TCconcentration and its effects on cell viability (MTT assay) weredetermined for the TCA dose range of 3–192 µg/mL. Based onthese experiments, 3T3-L1 mouse preadipocytes and adipocytes werepretreated for 4 h with or without 18 or 36 µg/mL of TCA followedby 18 h incubation with 200 ng/mL of lipopolysaccharide (LPS).Concentrations of secreted adipokines in the cell culture mediaand mRNA expressions were determined by ELISA and real-timepolymerase chain reaction, respectively. Western blot analysis wasconducted to determine activation of the nuclear transcription factorκB (NF-κB) pathway.

Results: Compared with the control group, in the rat serumsamples, there were no significant differences between the groups forthe monocyte chemoattractant protein 1, interleukin (IL)-6 and IL-10 concentrations. However, IL-6 mRNA expression in adipose tissuewas significantly (P < 0.05) reduced by the TCA supplementation. Inboth cultured 3T3-L1 preadipocytes and adipocytes, TCAs increasedcell viability and significantly (P < 0.05) reduced LPS-induced IL-6,tumor necrosis factor α (TNF-α), and toll-like receptors 3 and 4 (TLR-3and TLR-4) mRNA expressions. The anti-inflammatory effects of TCAwere mediated, in part, by reduction in NF-κB activity as indicatedby decreased LPS-induced p65 phosphorylation in TCA-treated cellscompared with control cells.

Conclusion: Tart cherry anthocyanins exert anti-inflammatoryeffects in rat adipose tissue and cultured adipocytes, which wasmediated, in part, by a reduction in NF-κB/p65 activity. Further clinicalstudies are warranted to test the efficacy of tart cherry supplementationin modulating adipose tissue inflammation in humans.

Funding SourcesObesity Research Cluster, Texas Tech University, Cherry Marketing

Institute.

Antioxidant Properties of Mint Essential Oils (P08-035)

Peng Ji, Zhaohai Wu, Bie Tan, and Yanhong Liu

University of California Davis

Objective: There is growing interest in identifying natural antiox-idant substance for use in animal feed and human food. The currentstudy aimed to evaluate the antioxidant properties of essential oils frompeppermint, spearmint, and scotch spearmint.

Methods: Three mint oils produced through a redistillation processwere evaluated for their antioxidant activity in vitro. The antioxidantproperties in terms of free radical scavenging, ferric iron reduction, andhydrogen donation were evaluated through 4 chemical-based assays,namely 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay(DPPH), Trolox equivalent antioxidant capacity assay (TEAC), reduc-ing power assay, and ferric reducing antioxidant power assay (FRAP).The inhibitory capacity ofmint oils against lipid peroxidationwas testedat various doses with the use of fresh pig liver homogenates with orwithout the presence of oxidants. Finally, a porcine jejunum epithelialcell line (IPEC-J2) was employed to determine the impact of addingmint oils at different concentrations on cellular antioxidant activity,and on the ratio of oxidized and reduced glutathione. All data wereanalyzed by PROC MIXED of SAS. The CONTRAST statement wasused to assess linear or quadratic effects of mint oils given at variousdoses.

Results: Mint oils all exhibited potent antioxidant activity inchemical-based assays. Peppermint oil had the lowest (P < 0.05) half-maximal effective concentration in the DPPH and TEAC assays, andthe highest reducing capacity in the FRAP and reducing power assaysas compared with the other two mint oils. All mint oils displayedinhibitory effects on lipid peroxidation in a dose-dependent manner(linear, P < 0.001) with the lowest lipid peroxidation observed at 1000µg/mL. Mint oils significantly increased the death of IPEC-J2 when theconcentration in culture medium reached 200 µg/mL. The maximalcellular antioxidant activity was observed at 5 µg/mL for peppermintoil, and 100 µg/mL for spearmint oil and scotch oil. The addition of 25µg/mLof spearmint oil or scotch oil increased (P< 0.05) the productionof glutathione from H2O2-treated IPEC-J2 cells.

Conclusions: Three essential oils all demonstrated antioxidantactivities in both chemical- and cell-based assays. An appropriate doseof mint oil is critical to inhibit oxidative response without damage tocell viability.

Milk Phospholipids Reduce Serum and Hepatic Cholesterol inLow-Density Lipoprotein–Receptor Knockout Mice Fed a Western-Type Diet (P08-036)

Christina Jiang,GregoryNorris, Courtney L.Millar, andChristo-pher Blesso


Objective: We have previously shown that chronic feeding ofpurified milk sphingomyelin (SM), a phospholipid (PL) component ofmilk fat globule membranes, is protective against hepatic steatosis anddyslipidemia in high-fat diet–fed mice. However, it is unclear whetheringestion of the totalmilk PL fraction can have similar effects as purifiedSM. Therefore, LDLr–/– mice, which are known for their sensitivity to


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diet-induced hyperlipidemia, were fed high-fat, high-cholesterol dietswith and without added milk PL from butter serum, a dairy by-productthat is rich in PL and SM.

Methods: Male LDLr–/– mice (age 6 wk) consumed ad libitumeither a high-fat, high-cholesterol diet (CTL; 45% kcal from fat, 0.22%cholesterol added by weight; n = 15), or the same diet supplementedwith 1% milk PL (1% MPL; n = 15) or 2% milk PL (2% MPL; n = 15)added by weight from butter serum. All diets were adjusted to matchin energy density, macronutrient composition, and calcium content.After 14 wk on diets, mice were fasted for 6 h prior to blood and tissuecollection/processing when they were killed.

Results:Nodifferenceswere detected in food intake, bodyweight, ortissue weights between the groups. Total serum cholesterol, non-high-density lipoprotein cholesterol, and nonesterified fatty acids (NEFAs)were significantly lower in the 2% MPL group than in the CTL group(−51%, −56%, and −26% respectively, P < 0.01). Serum monocytechemoattractant protein 1 (MCP-1) concentrations were decreasedby 56% in 2% MPL compared with CTL (P < 0.05). Hepatic totalcholesterol concentrations were reduced by 53% (P < 0.05) and 55%(P < 0.01) in the 1% and 2% MPL groups, respectively, compared withCTL. Hepatic cholesteryl ester concentrations were also decreased by67% (P < 0.05) and 74% (P < 0.05) in the 1% and 2% MPL groupscompared with CTL. The 1% MPL group had a 23% reduction inhepatic free cholesterol compared with the CTL group (P < 0.05). Nodifferences in hepatic triglycerides or phospholipids were found amongthe groups.

Conclusion: Milk PL demonstrated hypolipidemic effects bystrongly reducing serum and hepatic cholesterol pools in LDLr–/– micefed a Western-type diet. Milk PL also reduced the serum inflammatorymarker, MCP-1. Dairy by-products rich in SM, such as butter serum,could be utilized as value-added sources of milk PL to potentiallyimprove hyperlipidemia-related disease outcomes.

Funding SourcesThisworkwas supported by a research grant toCB from theNational

Dairy Council.

Effect of a Blend of Xylo-Oligosaccharides and Green CoffeeExtract on the Gut Microbiota Composition and Activity as Studiedin the SHIMEModel (P08-037)

T Alan Jiang,1 Pieter Van den Abbeele,2 Iris Pinheiro,2 andMassimo Marzora3

1Plexus Worldwide; 2ProDigest; and 3University of Ghent, Belgium

Objectives: The aim of this work was to evaluate the effect ofa combination of xylo-oligosaccharides (XOSs) and a polyphenolblend containing green coffee extract (SLIM, Plexus Worldwide) onmodulation of the microbiota composition and metabolism as well ason the gut wall activity.

Methods: A validated in vitro Human Intestinal Microbial Ecosys-tem (SHIME) model, coupled with co-cultures of epithelial cells andmacrophages, was used to investigate the prebiotic potential andsubsequent immunemodulation of long-term administration of a blendcontaining XOSs and polyphenols. The experimental setup of theSHIME run included a 2-wk start-up period, a 2-wk control period, anda 3-wk treatment period.

Results: SLIM was well fermented along the entire colon, leading toincreased production of butyrate (+7 mmol/L, 58%) in the ascendingcolon (AC), of acetate, propionate, and butyrate (∼+4 mmol/L each)in the transverse colon (TC), and of propionate (+4.1 mmol/L)in the descending colon (DC). The treatment also led to a milddecrease in ammonium (−7.5%) and increased lactate (55.5%) levelsin the AC. Health-related lactic acid bacteria such as Lactobacilliand Bifidobacteria were stimulated (∼+2.5 log/mL or 365-fold, and+2.1 log/ml or 290-fold, respectively) in the lumen. Additionally,Akkermansia muciniphila increased in the lumen of the proximal colon(+2 log/mL, or 257-fold). SLIM also showed a protective effect on theCaco-2 barrier function in the distal colon (TC and DC), for whicha significant increase (+18.5%) in transepithelial electrical resistancewas observed, an indicator of reduced permeability. Finally, uponfermentation, SLIM was found to have pronounced immune-activatingproperties in vitro (e.g., increase of nuclear transcription factor κBactivity).

Conclusions: SLIMwaswell fermented along the entire length of thecolon and showed potential prebiotic activities, being able to modulatethe activity and composition of the gut microbiota. This fermentationprofilewas correlatedwith a protective effect onCaco-2 barrier functionand pronounced immune-activating properties in the distal colon.

Funding SourcesThis study was funded by Plexus Wordwide.

Antiobesity Effects of Mulberry Fruit Powder Extracted withHigh Hydrostatic Pressure and Enzyme Hydrolysis in Rats Fed aHigh-Fat Diet (P08-038)

Sunyoon Jung, Mak-Soon Lee, Hyunmi Ko, Eunyoung Lee,Soojin Lee, Chaemin Kim, and Yangha Kim

Ewha Womans University, South Korea

Objectives: The aim of this study was to investigate the antiobesityeffect of mulberry (Morus alba L.) fruit powder extracted with highhydrostatic pressure and enzyme hydrolysis in rats fed a high-fat diet.

Methods: Sprague-Dawley rats (4 wk old, male) were randomlydivided into 4 groups and fed either a low-fat diet (LFD, 10% kcal fat),high-fat diet (HFD, 45% kcal fat), HFD + 0.5% mulberry fruit powderdiet (HML), or HFD + 1.0% mulberry fruit powder diet (HMH) for14 wk.

Results: Fourteen weeks of HML and HMH lowered the final bodyweight, body weight gain, and visceral adipose tissue mass (perirenal,mesenteric and total), comparedwith theHFDgroup (P< 0.05). On theother hand, the food intake and food efficiency ratio were not affectedby the HML and HMH. The levels of hepatic total lipid, triglyceride(TG) and total cholesterol (TC) were lower in the HMH group thanin the HFD group (P < 0.05). In addition, the hepatic TC level inthe HML group was lowered when compared with the HFD group(P < 0.05). Both HML and HMH reduced the serum levels of TG, TCand low-density lipoprotein cholesterol, and atherogenic index (AI),compared with the HFD group (P< 0.05). Furthermore, themRNA ex-pression of adipogenic genes such as peroxisome proliferator–activatedreceptor-γ (Ppar-γ ), sterol regulatory element–binding transcriptionfactor-1c (Srebp-1c), and fatty acid–binding protein 4 (Fabp4) was


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downregulated in the epididymal adipose tissue of rats in the HML andHMH group, compared with the HFD group (P < 0.05).

Conclusions: These results indicate that mulberry fruit powderextracted with high hydrostatic pressure and enzyme hydrolysis wouldhave beneficial effects on HFD-induced obesity, possibly throughinhibition of mRNA expression of genes related to adipogenesis.

Ginger Exerts an Antiarthritic Effect by Modulating Inflamma-tory Cytokine Levels in Collagen-Induced Arthritis: An In VivoMODEL (P08–039)

Vijaya Juturu,1 Cemal Orhan,2 Mehmet Tuzcu Tuzcu,2 NurhanSahin,2 Suleyman Koca,2 and Kazim Sahin2

1OmniActive Health Technologies Inc.; and 2Firat University,Turkey

Objective: Rheumatoid arthritis is a chronic autoimmune diseasecharacterized by an increased synovial inflammatory response due todestruction or erosion of articular cartilage in major joints. Gingerextract—high-potency ginger with 30% bioactives and a minimumof 25% gingerols—has antioxidant and anti-inflammatory properties.The aim of this study was to investigate the effect of ginger extracton oxidative stress and inflammatory responses in a collagen-inducedrheumatoid arthritis (CIA) rat model.

Methods: Female Wistar rats (n = 7; 8 wk old; 180 ± 20 g) wereallocated to 3 groups: Group I rats were treated with saline and served asa control; Group II rats were subjected to CIA by intradermal injectionof bovine collagen II type and served as the induced group; and GroupIII rats were subjected to CIA and given 50 mg/kg body wt gingerextract (Gingever) for 33 d. Decreased arthritic scores, decreased serumlevels of interleukin (IL) 6, IL-17, tumor necrosis factor α (TNF-α),and dickkopf-related protein 1 (DKK-1), and increased serumobestatinand sclerostin levels were observed in Group III animals. Group IIIshowed reduced levels of TNF-α, IL-17, nuclear factor κB (NF-kB), andcyclooxygenase 2 (COX-2) protein levels in joint tissues. A significantdecrease in inflammation anddestruction scoreswas observed inGroupIII compared with Group II treatment.

Conclusion: The results of this study show that ginger extracttreatment (Group III) may affect the inflammatory cascade in CIA ratsand may show antirheumatoid arthritis properties.

Funding SourcesOmniActive Health Technologies Ltd. India.

Blockade of High Glucose–Induced Glomerular Fibrosis byTangeretin through Inducing Autophagy (P08-040)

Min kyung Kang, Jung Han Yoon Park, and Young-Hee Kang

Hallym University, South Korea

Objectives:Diabetic nephropathy is one of the most important dia-betic complications prompted by chronic hyperglycemia, characterizedby glomerulosclerosis and tubular fibrosis, eventually causing kidneyfailure. An emerging body of evidence suggests that targeting the au-tophagic pathway to restore autophagy activity may be renoprotective.Tangeretin is a polymethoxyflavone present in tangerine and othercitrus peels, and has anticancer and anti-inflammatory effects. Thisstudy explored the involvement of autophagy in glomerular fibrosis, and

investigated effects of tangeretin on glomerular fibrosis and autophagyrestoration through inhibition of the PI3K/AKT/mTOR pathway.

Methods: Human renal mesangial cells were exposed to 33 mMglucose in the absence and presence of 1–20 μM tangeretin for upto 6 d. Cellular expression levels of the fibrogenic collagen-I and theautophagy-related proteins of beclin-1, LC3, P62/SQSTM1, and VPS34were examined. In addition, PI3K, AKT, and mTOR gene proteins werescreened to reveal the relationship betweenPI3K/AKT/mTOR signalingpathway, autophagy, and glomerular fibrosis.

Results: High glucose promoted the production of collagen-1 inmesangial cells, which was dose-dependently inhibited by 1–20 μMtangeretin. Western blot data showed that high glucose elevated theexpression of the autophagy marker beclin-1 and autophagosomeformation protein LC3 in a temporal manner. When tangeretin wassupplemented tomesangial cells exposed to high glucose, the expressionof beclin-1, LC3, and P62/SQSTM1 was dampened. In addition,the autophagy-specific VPS34 was downregulated by administeringtangeretin to mesangial cells stimulated by high glucose. Finally, PI3K,AKT, and mTOR were activated in high glucose–exposed mesangialcells, and such activation was encumbered by tangeretin.

Conclusions: These results demonstrated that tangeretin bluntedglomerular fibrosis and restored autophagy via inhibition of thePI3K/AKT/mTOR pathway. Tangeretin may be a potent renoprotectiveagent counteracting diabetes-associated glomerular fibrosis leading tokidney failure.

Funding SourcesThis work was supported by the National Research Founda-

tion of Korea grant funded by the Korea government (NRF-2017R1A6A3A04011473).

Anti-Hepatitis Effect ofKoreanThistle onCarbonTetrachloride–Induced Hepatitis Rat (P08-041)

Soon Ah Kang,1 Ji-Hae Kim,1 and Kun-Young Park2

1Hoseo University, South Korea; 2Cha Univerisity, South Korea

We investigated the hepatitis inhibition effect of Korean thistleextracts on rat hepatitis induced by carbon tetrachloride (CCl4).We divided the animals into seven groups: normal (no treatment),control (added CCl4), C-T-L (CCl4 + thistle low concentration), C-T-M (CCl4 + thistle medium concentration), C-T-H (CCl4 + thistle highconcentration), C-AC (CCl4 + l-acetylcystine), and C-S (CCl4 + sily-marin). The weights of the animals in each group and the weightsof organs (kidney, spleen, testes) did not show any signigificantdifference. However, liver weight was increased in the CCl4-treatedgroup compared with the normal group. Serum levels of ALT andAST were significantly higher in the control group than in the normalgroup, but ALT and AST levels in the C-T-H group were significantlylower than in the control group (P < 0.05). Lactate dehydrogenase(LDH), another liver enzyme, was found to be higher in the controlgroup than in the normal group. LDH levels in the C-T-H groupwere also significantly lower than in the control group (P < 0.05).In addition, the C-T-H group showed no significant difference fromthe positive control group. Serum triglyceride (TG) was higher in thecontrol group than in the normal group, but the serum TG of theC-T-H group was significantly lower than that of the control group


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(P < 0.05). Total cholesterol was also significantly increased in thecontrol group compared with the normal group. In the liver, catalaseand superoxide dismutase (SOD) activities were decreased in the CCl4-treated group compared with the normal group, and SOD activity wassignificantly increased in the C-T-H group compared with the controlgroup (P < 0.05). Therefore, a high intake of thistle extract by ratswithCCl4-induced hepatitis decreases the level of liver enzymes, neutrallipids in serum, and increases SOD activity in liver. This suggests thatthe antioxidant effect of thistle extract suppresses the inflammatoryresponse in the liver, and the C-T-H group has a similar effect to thepositive control, which is considered to be effective for antioxidant andhepatitis suppression of thistle extract.

Anticancer Effects of Commercial Doenjangs in Azoxymethane/Dextran Sulfate Sodium–Induced Colitis-Associated Colon Cancerin C57BL/6J Mice (P08–042)

Soon Ah Kang,1 Sung-Eun Choi,1 and Kun-Young Park2

1Hoseo University, South Korea; and 2Cha University, South Korea

This study was conducted to investigate the anticancer effects ofcolorectal cancer in C57BL/6J mice. In order to confirm the anticancereffect of doenjang (soybean paste) on colorectal cancer, the mice weredivided into 7 groups: normal control group (Nor), control group(Con), miso doenjang from Japan diet group (M-D), Korean traditionaldoenjang diet group (T-D), home-made doenjang diet group (H-D),complex doenjang diet group (C-D), and Korean doenjang diet group(K-D). After the end of the experiment, the length/weight ratio of thecolon was measured, and in the C-D and K-D groups this ratio wassignificantly lower than in the Con group (P < 0.05). The numberof tumors in the colon was not significantly different in the M-D, T-D, and H-D groups compared with the Con group, but the numbersof tumors in the C-D and K-D groups was significantly reduced(P < 0.05). Histopathologic analysis of the colon revealed that theC-D and K-D groups had recovered intestinal tissue similar to thatof the normal control group, compared with the Con group, whichhad high tumor development and cancer cell infiltration. In case ofserum proinflammatory cytokines, tumor necrosis factor α levels weresignificantly reduced in the C-D and K-D groups compared with inthe Con group, and the interleukin 1β and interleukin 6 levels werealso significantly reduced (P < 0.05). The mRNA levels of iNOS andCOX-2 identified by reverse transcriptase-polymerase chain reactionwere significantly reduced in all experimental groups compared withtheCon group (P< 0.05). In addition,mRNAexpression levels of cyclinD1were significantly reduced in all experimental groups comparedwiththeCon group, and caspase-3 and caspase-9were significantly increasedin the C-D and K-D groups compared with the Con group (P < 0.05).The K-D and C-D diet groups have higher anticancer effects in themice with induced colon cancer compared with the other experimentalgroups. It seems that the anticancer effect was induced by the traditionalmeju (dried fermented soybeans) used in both these groups, and thisdifference is considered to be due to the materials or manufacturingmethods used by the other groups.

Physicochemical Characteristics of Jangachi-Added Angelica gi-gasN. (P08-043)

Soon Ah Kang and Myo Jung Kim

Hoseo University, South Korea

This study was conducted to investigate potential health benefits ofpickled medicinal crops (jangachi) by examining the physicochemicalcharacteristics of jangachi-added Angelica gigasN. The jangachi-addedA. gigas N. had a total polyphenol content of 0.17 μM quercetinequivalent per 100 g ofA. gigasN.TheDPPH radical scavenging activityand the superoxide dismutase activity of A. gigas N. increased withincreasing concentration of A. gigasN. Analysis of the physicochemicalproperties of jangachi supplemented with A. gigas N. showed that thebrightness increased as the amount of A. gigas N. extract increased.The pH of jangachi-added A. gigas N. was the lowest when 6 g of A.gigas N. was added; the highest salinity was obtained when 6 g of A.gigas N. was added; and highest sugar contents were obtained with 3and 6 g of A. gigas N. The results of this study suggest that the valueof medicinal crops will be improved by activating these crops withjangachi. This approach should broaden the availability of medicinalcrops and increase the added value of utilizing fermented food. Wepredict that addition of jangachi will be useful as a basic method toimprove the utility value of medicinal crops.

A Randomized, Double-Blind, Crossover, Placebo-ControlledStudy on Preventive Effects, Safety, and Efficacy of Amla (EmblicaofficinalisGatertn.) in Healthy Human Subjects (P08-044)

Mahendra Parkash Kapoor,1 Tsutomu Okubo,1 and Koji Suzuki2

1Taiyo Kagaku Co. Ltd., Japan; and 2Suzuka University of MedicalScience, Japan

Objective:Amla (Emblica officinalisGaertn.), an edible Indian fruit,has been used in traditional Ayurvedic medicinal formulations. Weinvestigated the preventive effect of oral administration of a standard-ized formulation of amla supplementation (SunAmla-PD1) on vascularfunction, blood hematology, glucose and lipid profiles, oxidative andinflammatory biomarkers, urinalysis, and liver hepatotoxicity safety inadult healthy humans.

Methods: Fourteen subjects aged 36–67 y completed the 18-wkstudy, during which they received placebo and SunAmla-PD1 (500mg/d) according to study protocols. Differences during the study periodwere identified (P ≤ 0.05) through the use of a repeated-measureANOVA for within-group effect. Group differences were identified(P ≤ 0.05) by ANCOVA at each sampling time point.

Results: The results indicate that SumAmla-PD1 improvesblood fluidity, lowers the von Willebrand factor, and reduces 8-hydroxydeoxyguanosine and soluble thrombomodulin concentration,along with a significant increase in HDL-cholesterol and reductionin LDL-cholesterol. No meaningful, significant abnormal clinicalchanges could be observed after SumAmla-PD1 intake on hematology


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or liver hepatotoxicity or urinalysis examinations, confirming thesafety of SumAmla-PD1. Furthermore, no adverse events inducedby the test supplement were observed throughout the studyduration.

Conclusion: Oral intake of SunAmla-PD1 has good acceptabilityand is safe for consumption as part of a healthy lifestyle.

Funding SourcesFunded by Taiyo Kagaku and Suzuka University.

N-Acetylcysteine Treatment against Respiratory and CardiacMuscle Abnormalities Induced by a High-Fat, High-Sugar Diet inRats (P08-045)

Rachel C Kelley, Derek Muscato, Ravi A Kumar, Demetra DChristou, and Leonardo F Ferreira

University of Florida

Objective: Western diets [high-fat, high-sugar (HFHS)] induceobesity-related health concerns that arise, in part, from respiratoryand cardiovascular dysfunction. Respiratory muscle abnormalitiesmay contribute to impaired breathing in obese individuals. Excessreactive oxygen species (ROS) cause respiratory and cardiac muscledysfunction. Thus, we tested if treatment with an antioxidant nutri-tional supplement, N-acetylcysteine (NAC), prevents respiratory andcardiac muscle pathophysiology in rats consuming a Western/HFHSdiet.

Methods: Adult male Wistar rats consumed ad libitum lean (20%protein, 70% carbohydrate, 10% fat kcal) or HFHS (20% protein, 35%carbohydrate, 45% fat kcal) diets for∼22 wk. HFHS rats were randomlyallocated to control orNAC (2mg/ml) in drinkingwater for the last 8wk(3 groups: lean, HFHS, and HFHS-NAC; n = 8 per group). Respiratory(diaphragm) and cardiac muscles were evaluated through the use ofmorphology and standard physiologic assays (i.e., in vitro contractileanalyses and echocardiography). Data are mean ± SE, ∗P < 0.05compared with other groups.

Results: NAC prevented the excess weight gain induced by theHFHS diet (body weight in grams: lean 597 ± 22, HFHS 693 ± 25∗,HFHS-NAC 528 ± 22). Surprisingly, the HFHS diet did not decreasediaphragm maximal force (in N/cm2: lean 29 ± 0.7, HFHS 28 ± 1.1,HFHS-NAC 26 ± 0.8) or peak power (in W/kg: lean 263 ± 12,HFHS 250 ± 9.2, HFHS-NAC 254 ± 19.5). NAC prevented cardiachypertrophy in HFHS rats (heart weight in mg: lean 1118 ± 31, HFHS1314 ± 50∗, HFHS-NAC 1162 ± 46), and restored to normal indicesof left ventricle diastolic, e.g., E wave deceleration rate (in m/s2: lean22 ± 1.0, HFHS 27 ± 1.1∗, HFHS-NAC 20 ± 1.3) but not systolicfunction, e.g., fractional shortening (in %: lean 44 ± 2∗, HFHS 54 ± 3,HFHS-NAC49± 2). Analyses of ROS emission and glutathione contentare ongoing.

Conclusions: Western diet did not compromise diaphragm musclefunction, suggesting nonmuscle contributors to breathing abnormali-ties in obesity. Importantly, NAC prevented cardiac hypertrophy anddiastolic dysfunction induced by Western diet. Our initial findingssuggest that NAC supplementation may be beneficial in the clinicaltreatment of obesity.

Funding SourcesT32 HL134621.

Fast Absorption of Amino Acids fromWhey Protein HydrolysateCompared withWhey Protein Concentrate (P08-046)

Nathaniel Y Kerr,1 Jordan Joy,1 Nancy M DiMarco,1 Shane KBroughton,1 Robert ECWildman,1 Ulla Mikkelsen,2 RoxanneMVogel,1 and John Beatty1

1Texas Woman’s University; and 2Arla Foods

Objective: Whey is a popular source of supplemental protein dueto the completeness and quantity of branched-chain amino acids(BCAAs). Protein digests into amino acids (AA) at different rates,affecting delivery to, and actions within, peripheral tissues. Currently,few studies have compared plasma appearance of amino acids fromwhey protein hydrolysate (WPH) with intact whey protein concentrate(WPC). Therefore, purpose of the investigation was to determineplasma AA appearance betweenWPH,WPC, and carbohydrate (CHO)control supplements in healthy males based on the hypothesis thatWPH will elicit greater elevations in circulating AAs than WPC.

Methods: In a double-blind crossover design, 15 recreationallyactive males consumed 0.3 g of macronutrient/kg lean soft tissue asWPH (Lacprodan Hydro.365, degree of hydrolysis, 26; Arla Foods),WPC (LacprodanWPC80; Arla Foods), or maltodextrin (CHO). Bloodwas collected before and at 0, 10, 20, 30, 45, 60, 90, 120, and 180 minfollowing ingestion. Plasma was extracted and frozen at −80°C untilanalysis. Samples were derivatized (Phenomenex, Torrance, CA) andanalyzed by GC-MS. GC-MS chromatograms were analyzed for AApresence and abundance. Data are reported as mean ± SD.

Results: Significantly (P < 0.01) greater plasma appearance of allBCAAs was observed for WPH (+2023.2 au) and WPC (+1558.2 au)than CHO (−580.5 au) with no difference between proteins over 180min. During first 20 min, plasma appearance of isoleucine was greater(P < 0.001) in WPH (+86.1 au) and WPC (+53.8 au) treatments thanCHO (−2.5 au). During first 20 min, leucine plasma appearance fromWPH (+86.1 au) was significantly greater than WPC (+60.1 au) andboth had significantly greater appearance than CHO (+1.2 au). Valineplasma appearance fromWPH (+123.4 au) was significantly (P< 0.05)greater than WPC (−54.6 au) after 30 min and both were significantlydifferent from CHO (+20.7 au). During the first 20 min, total BCAAplasma appearance from WPH (+334.2 au) was significantly greaterthan WPC (+59.2 au) and CHO (+19.5 au)

Discussion: Amino acid plasma appearance was significantlyincreased after protein consumption when supplementing WPH com-pared with WPC. Recreationally active males may benefit more fromconsumption of WPH rather thanWPC to stimulate muscle repair andmaintenance.

Funding SourcesAuthors gratefully acknowledge financial contributions and product

from Arla Foods Ingredients Group P/S, Viby, Denmark.

Effects of Ethanolic Extract Steamed Ginger (Zingiber officinale)in Diet-Induced Obesity (P08-047)

Bohkyung Kim, Hee-Jeong Kim, and Youn-Soo Cha

Chonbuk National University, South Korea

Objective: The potential effects of ginger (Zingiber officinale), aroot vegetable, on prevention of obesity is well known. Root vegetables


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are reported to change their chemical profiles and bioactivities whensteamed or heated. In the present study, the antiobesity effects of anethanolic extract of steamed ginger in diet-induced obese mice wereinvestigated.

Methods: Male C57BL/6J mice were fed a normal diet (ND,10% wt/wt fat), high-fat diet (HFD, 60% wt/wt fat, n = 10), HFDsupplemented with either 40 or 80 mg/kg of GSE (GHFD4 or GHFD8,respectively) for 12 wk (10 mice/group). Serum lipids, glucose, insulin,adiponectin, and leptin were measured. The expression of genesinvolved in adipogenesis and lipid metabolism were measured inepididymal adipose tissue. Histologic analysis of adipose tissue wasperformed.

Results: HFD significantly increased the body weight, liver andepididymal adipose tissue weights compared with ND control, whereasGSE supplementation attenuated the increases by HFD. The increasesin serum total cholesterol, LDL cholesterol, triglyceride, glucose, andinsulin by HFD were significantly decreased in GHFD4 and GHFD8.The mRNA expression of adipogenesis and lipid metabolism relatedgenes were significantly altered by HFD. However, GSE significantlydecreased the expression of genes for adipogenesis and lipogenesis suchas peroxisome proliferator-activated receptor γ , CCAAT-enhancer-binding proteins, aP2, sterol regulatory element-binding protein 2, fattyacid synthasem and acyl-coA carboxylase in epididymal adipose tissue.Moreover, the size and the number of adipocytes were significantlydecreased in adipose tissue of GHFD4 and GHFD8 compared withthose of HFD.

Conclusions: The present study indicates that an ethanolic extractof ginger has antiobesity potential by decreasing the adipose tissue sizethrough the regulation of adipogenesis and lipid metabolism relatedgenes in the epididymal adipose tissue of diet-induced obese mice.

Eucalyptol Blocks Diabetes-Associated Loss of Glomerular SlitJunctions and Podocyte Foot Process Effacement (P08-048)

Dong Yeon Kim and Young-Hee Kang


Objective:Prolonged hyperglycemia in diabetic nephropathy causesglomerular podocyte damage that can lead to podocyte loss andfoot process effacement. Eucalyptol (1,8-cineole) is a natural organicessential oil which is a monoterpenoid present in eucalyptus oil withanti-inflammatory and antioxidant properties. This study investigatedthe renoprotective effects of eucalyptol on podocyte slit junctions andfoot process effacement under a diabetic ambience.

Methods: Immortalized mouse podocytes were incubated in mediacontaining 33 mM glucose or 100 µg/mL AGE for 4 d in the presenceof 1–20 μM eucalyptol. Antibodies of nephrin, podocin, integrin β1,α-actinin-4, CD2AP, advanced glycation end products (AGEs) andreceptor for AGEs (RAGE) were used for Western blot. The in vivoanimal model involved oral administration of 10 mg/kg of eucalyptol.Extracts of tissues cut down to 5 µM thickness were Western blottedor analyzed by immunohistochemistry. Kidney tissue sections wereobserved by transmission electron microscopy (TEM).

Results: Eucalyptol-enhanced expression of the podocyte slit di-aphragm proteins of nephrin, podocin and integrin β1 was down-regulated by glucose stimulation. In addition, eucalyptol restored the

expression of CD2AP, FAT1 and α-actinin-4. The RAGE inductionwas elevated by exposure of podocytes to high glucose and AGEs,which was reduced by eucalyptol in a dose-dependent manner.TEM revealed that podocyte foot process effacement occurred indiabetic mouse glomeruli. However, oral administration of eucalyptolimproved foot process effacement of diabetic podocytes by inhibitingthe loss of glomerular slit junction proteins CD2AP and α-actinin-4. Increased induction of AGEs and histologic alterations in diabetickidney tissue declined in the eucalyptol-treated mice, evidenced byimmunohistostaining and periodic acid Schiff staining.

Conclusions: These results demonstrated that eucalyptol main-tained normal podocyte foot process and slit diaphragm structures.Therefore, eucalyptol may be a potent renoprotective agent combat-ing diabetes-associated podocyte dysfunction and maintaining theglomerular filtration barrier.

Funding SourcesThis work was supported by the National Research Founda-

tion of Korea grant funded by the Korea government (NRF-2017R1A6A3A04011473).

Piceatannol Modulates Proteomic Profile of Proteasome and itsActivity (P08-049)

Kee-Hong Kim,1 Xiaoxuan Guo,2 Jordan MKI Oshiro,1 PeiyiShen,3 Ou Wang,3 and Yeonhwa Park3

1Purdue University, IN; 2China Agricultural University; 3Universityof Massachusetts

Objectives: Piceatannol is a natural analog of resveratrol thatmodulates various cellular events such as oxidative stress, inflammation,cell proliferation, and energy metabolism. However, the systemicimpact of piceatannol and its target cellular pathways in physiologicconditions remain elusive. Our recent proteomics study revealed thatpiceatannol, unlike resveratrol, promotes the proteomic profiles relatedto proteasome catabolism in Caenorhabditis elegans. To further explorethe functional implication of piceatannol in proteasome catabolismin mammalian cells, this study determines the effect of piceatannolon proteasome activities and the expression of genes involved inproteasome assembly in human HeLa cells.

Methods: Cells were treated with piceatannol (12.5 μM) for 24 h.Proteasome fraction and total RNA were purified from these cells toexamine the effect of piceatannol on trypsin-like, chymotrypsin-like,and caspase-like activities through the use of their specific fluorogenicpeptide substrates, and the expression of the proteasome genes by real-time PCR assay.

Results: We identified that piceatannol treatment resulted in a30% increase in trypsin-like proteasome activity (P = 0.042) and a75% increase in chymotrypsin-like proteasome activity (P = 0.044),with no significant effect on the caspase-like proteasome activity whencompared with non-treated controls. However, piceatannol exhibitedlittle effect on mRNA levels of genes involved in α- and β-typeproteasome subunits, ATPase and non-ATPase subunits of 19S, and the11S regulator in HeLa cells.

Conclusion: Collectively, our data indicate that piceatannol fa-cilitates proteomic profiles of proteasome and its activity in a genetranscription–independent manner.


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Funding SourcesCore Pilot Funding from IndianaClinical andTranslational Sciences

Institute (UL1TR001108).

Anti-Inflammatory Effects of Cinnamon in a Mouse Model ofDextran Sulfate Sodium–Induced Colitis (P08-050)

Kyeong Jin Kim,1 Ji Sun Youn,1 Min Seo Kim,1 Ji Yeon Kim,1 andYeon Seok Kim2

1Seoul National University of Science and Technology, South Korea;2Chunho Bio

Objective: This study aimed to evaluate the anti-inflammatoryeffects of cinnamon on colitis induced by 5% dextran sulfate sodium(DSS) in BALB/c mice.

Methods: The experimental animals were divided into 5 groups:control, DSS-induced colitis, and 100 300, and 500 mg cinnamon/kgbody wt. The mice were fed distilled water or cinnamon extracts oncea day for 21 d. Colitis was induced from day 14 to 21 by administrationof 5% DSS in drinking water. We evaluated the change in weight, colonlength, and the pathologic disease activity index (DAI).

Results: Compared with the mice in the DSS group, those inthe cinnamon groups showed an increase in body weight at 21 d.The cinnamon extracts significantly decreased the shortening of thecolon. The length of the colon of mice receiving 500 mg/kg cinnamonincreased to 9.8 cm, whereas that of the mice receiving DSS was 8.0 cm.ThemeanDAI in the cinnamon groupswas significantly lower than thatin the DSS group from day 4. Administration of 500 mg/kg cinnamonwas associated with the highest decrease in DAI, by 0.375 points.

Conclusions: Our results indicate that cinnamon has protectiveeffects against DSS-induced colitis. Therefore, we conclude that cinna-mon may be used for the prevention and treatment of irritable bowelsyndrome.

Funding SourcesThis work 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 DevelopmentProgram, funded by theMinistry of Agriculture, Food and Rural Affairs(MAFRA) (project no. 116012-3).

Inhibition of Osteoclast Activation by Korean Thistle (Cirsiumsetidens Nakai) Ethanol Extracts in RANKL-Differentiated MurineMacrophages (P08-051)

Soo-il Kim and Young-hee Kang


Objectives: Bone-remodeling imbalance induced by increasedosteoclast formation and bone resorption is known to cause skeletaldiseases such as osteoporosis. Botanic antioxidants are now beingincreasingly investigated for their health-promoting effects on bone.Korean thistle, a wild perennial, is widely consumed as a food andtraditional medicine in Korea with anti-inflammatory and antioxidantactivities due to the flavone-type flavonoids luteolin and apigenin. Thisstudy investigated whether 10–50μg/mL Korean thistle extract inhibitsosteoclast activation in RANKL-differentiated murine macrophages.

Methods: This study investigated Korean thistle RANKL-induced osteoclast differentiation and fusion in RAW 264.7 murinemacrophages incubated with≤20 μg/mL Korean thistle extract for 5 d.

Results: These extracts inhibited the RANKL-induced multinu-cleated osteoclast formation by suppressing tartrate-resistance acidphosphatase induction and its activity. In addition, the treatment ofmurine macrophages with 20 μg/mL Korean thistle extract reducedcellular expression of carbonic anhydrase II and integrin β3 elevated byRANKL. When Korean thistle extracts were administered to RANKL-exposed macrophages, bone-resorbing activity was highly attenuated.On the other hand, 20 μg/mL Korean thistle extract increased alkalinephosphatase activity in osteoblastic MC3T3-E1 mouse cells, indicatingthat the extract enhanced osteoblast differentiation.

Conclusions: Korean thistle is a natural therapeutic agent thatimproves osteogenesis.

Gamma-Tocotrienol Attenuates Hepatic Steatosis and Endoplas-mic Reticulum Stress against High-Fat and High-Sucrose Diet (P08-052)

Yongeun Kim1 and Soonkyu Chung2

1Nutrition and Health Sciences; 2University of Nebraska-Lincoln

Objective: Gamma-tocotrienol (gT3), an unsaturated isomer of vi-tamin E, has been reported to exhibit antiobesity and anti-inflammatoryproperties against high-fat (HF) diets. This study aims to define therole of gT3 in the pathogenic progression of hepatic steatosis andfibrosis with a combined dietary stimulus of HF and chronic sucroseconsumption (HFHS).

Methods: To promote hepatic stress, obesity-prone C57BL/6 malemice (n = 8/group) were challenged with an HFHS dietary regimencomposed of 20% sucrose drink and HF (45% calories from fat) alone(HFHS) or HF-diet containing 0.1% of gT3 (HFHS-gT3). Mice few alow-fat diet (LF, 10% calories from fat without sugar intake were used asthe control. After 16 wk, hepatic steatosis was quantified by triglyceride(TG) content and lipogenic gene expression by quantitative polymerasechain reaction. The markers of hepatic toxicity, plasma lipid profiles,were quantified by chemical analysis. The endoplasmic reticulum (ER)stress markers and signaling were determined byWestern blot analysis.The impacts of gT3 on energy expenditure and insulin sensitivity weremeasured by metabolic cage and by glucose and insulin tolerance test,respectively.

Results: Chronic HFHS challenge induced massive weight gain andenlargement of liver compared with LF, which was significantly reducedin mice fed a HFHS-gT3 diet without altering food intake. Comparedwith mice fed HFHS alone, HFHS-gT3 fed mice featured substantialreductions of 1) hepatic steatosis evidenced by ∼50% decrease ofhepatic TG content and lipogenic gene expressions including Srebp1c,Dgat2, Fas, Scd1, and Lpl, 2) hepatic ER stress by lowering thesignaling of p-JNK, CHOP, BiP, and p-eIF2a, and 3) hepatic toxicityby decreasing alanine aminotransferase and aspartate aminotransferaselevels, inflammatory gene expression, and expression of the fibrosis-related gene, Timp1. In addition, the presence of gT3 in HSHS wasassociated with increased whole-body energy expenditure, improvedinsulin sensitivity, and reduced adipose and pancreatic inflammationcompared with the dietary stimulus of HFHS only.


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Conclusions: Our results demonstrated that gT3 supplementationwas effective in attenuating HFHS diet–mediated hepatic steatosis andER stress. This work suggests that inclusion of gT3 in a diet mayhelp mitigate or prevent nonalcoholic fatty liver diseases and fibrosisprogression.

Funding SourcesThis studywas supported byAmericanHeartAssociation SDGgrant

to SC (13SDG14410043), the USDA-Hatch grant at the University ofNebraska-Lincoln.

Leucrose, an Isomer of Sucrose, Suppressed Dextran SulfateSodium–Induced Colitis in Mice by Regulating Anti-InflammatoryCytokines and Gut Bacteria of the Phylum Verrucomicrobia (P08-053)

Yuri Kim,1 Eunju Kim,1 Jihye Lee,1 Daeyeon Lee,2 and Sang-HoYoo2

1Ewha Womans University, South Korea; and 2Sejong University,South Korea

Objectives: The incidence of inflammatory bowel disease (IBD)has been increasing. High sugar consumption has been proposedto be one of the risk factors for IBD. Leucrose (d-glucopyranosyl-α(1–5)-d-fructopyranose) is a natural sucrose isomer. The purposeof the present study was to investigate the anti-inflammatory effectsof leucrose in a dextran sulfate sodium (DSS)–induced colitis mousemodel.

Methods: Colitis was induced by treating mice with 2 cycles of DSS(2.5%) in drinking water for 5 d, with untreated water being suppliedfor 10 d between the DSS treatments. Leucrose was replaced at 25%or 50% of the total sucrose amount in the diet. Leucrose pretreatmentwas administered 2 wk before the DSS treatment and supplementedfor whole experimental period. Physiologic inflammation markers,histopathologic evaluation, inflammatory cytokines, and related signal-ing were analyzed. Microbiota were investigated through the use ofpyrosequencing in fecal samples.

Results: Disease activity index (DAI) scores, colon length,histopathologic damage, and MPO activity levels were improvedby leucrose supplementation. mRNA levels of proinflammatorycytokines, includingMCP-1 and TNF-α and protein levels of iNOS andCOX2 were downregulated in the leucrose supplementation group.Phosphorylation of proinflammation-relatedmitogen-activated proteinkinases (MAPKs) was reduced in the leucrose supplementation groupcompared with the colitis group. Leucrose supplementation did notaffect microbiota and diversity. However, the phylum Verrucomicrobiawas more abundant in the colitis group compared with the controlgroup whereas leucrose supplementation lowered the proportion ofVerrucomicrobia.

Conclusion: The result indicated that leucrose exerted an anti-inflammatory effect by improving colitis conditions through controllinginflammatory cytokines andMAPK signaling in colon and by regulatingbacteria of the phylum Verrucomicrobia.

Funding SourcesThis research was supported by National Research Foundation of

Korea (NRF-2015R1A2A1A15056119).

Arginase Inhibition by Inositol-StabilizedArginine Silicate (ASI;Nitrosigine): A Novel Mechanism by which ASI Enhances ArginineBioavailability (P08-054)

James Komorowski,1 Sara Perez Ojalvo,1 Sarah Sylla,1 and EmirVeledar2

1Nutrition 21, LLC; and 2Emory University, GA

Objectives: Several studies have shown that inositol-stabilizedarginine silicate (ASI; Nitrosigine) clinically enhances blood levelsof arginine and nitric oxide (NO), making it a popular ingredientin sports nutrition supplements. ASI increases plasma arginine by>70% compared with arginine hydrochloride (ArgHCl), and does sofor a significantly longer duration (up to 6 h), indicating that ASIis a more bioavailable form of arginine. However, the mechanismby which ASI enhances plasma arginine levels more than ArgHClis still unknown. This clinical trial was designed to explore thehypothesis that ASI enhances plasma arginine levels through theinhibition of arginase and asymmetric dimethylarginine (ADMA), asthese enzymes are known to inhibit arginine levels and NO synthesis,respectively.

Methods: To explore this hypothesis, human plasma samples weretested for arginase and ADMA levels after 15 d of supplementation withASI or ArgHCl. Ten healthy males per treatment group, aged 18–40 y,with body mass index ≥18.5 to <25 kg/m2, were randomly assignedto take a single daily oral dose of ASI or ArgHCl (each containing atotal of 500 mg of arginine) for 15 d. These subjects attended studyvisits on days 1 and 15, with a 7-d washout period between test productadministration. Fasting blood sampleswere collected before dosing, andat 0.5, 1, 1.5, 2, 3, 4, 5, and 6 h postdose for plasma arginine, arginase,and ADMAmeasurements.

Results: Study results show that after 15 d of supplementation,arginase levels were significantly different between the ASI and ArgHClgroups (P < 0.05). At multiple time points after supplementation,arginase levels were reduced by ASI, whereas there were no changesdetected after ArgHCl supplementation. ADMA levels did not differbetween the ASI and ArgHCl groups, and did not change comparedwith baseline.

Conclusions: The results of this study show that ASI supplemen-tation inhibits arginase levels, but not ADMA levels, whereas ArgHCldoes not significantly affect either enzyme. These results support thefindings that ASI supplementation, after single dose (1 d) and multipledoses (15 d), inhibits the metabolic pathway that catabolizes arginine,thereby enhancing arginine bioavailability, and, in turn, increases NOexpression.

Funding SourcesThis study was funded by Nutrition 21, LLC.

Content and Composition of Phenolic Compounds, and Antiox-idant Activity of Blueberries Grown in Korea (P08-055)

Eun-Jung Kwak,1 Kyung-Ah Kim,2 Jae-Hyeok Jang,1 and Young-Rok Choi1

1Yeungnam University, South Korea; and 2Chungnam NationalUniversity, South Korea


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Objectives: Blueberry belongs to the Ericaceous Vaccinium genusof deciduous shrub plants and is known for its high contents ofvarious bioactive compounds including flavonoids, phenolic acids,and anthocyanins. These phytochemical compounds are responsiblefor various health benefits of berries, such as antioxidant, antitumor,and anti-inflammatory properties. The present study was aimedto investigate the physicochemical properties, the content, and thecomposition of phenolic compounds, and the antioxidant activity of 10blueberry cultivars grown in Sangju, South Korea.

Methods: We evaluated the content of total phenolic compounds,total flavonoids, and total anthocyanins. The composition and contentof phenolic compounds were investigated by HPLC with diode arraydetection. Antioxidant activities were evaluated by ferric reducingantioxidant power (FRAP) and the free radical scavenging activ-ities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The cellular antioxidantactivities in HepG2 cells were measured with a DCFDA cellular ROSdetection assay kit.

Results: The highest levels of bioactive compounds and FRAP aswell as the free radical scavenging activities of DPPH and ABTS werefound in the Rubel cultivar, followed by the Rancocas and Sunrisecultivars, in vitro and in cellular systems. TheChandler cultivar bore thebiggest fruit, though these had very low bioactive compound contentsand antioxidant activities.

Conclusions: There were differences in the content and thecomposition of phenolic compounds, and antioxidant activity, amongthe different blueberry cultivars. These results will provide informationon the selection of breeding as well as for the development of dietarysupplements containing blueberries.

Grain Sorghum Bran Polyphenols and Gut Microbial Fermenta-tion Patterns (P08-056)

Wing Shun Lam, Danielle Ashley, Cindi Brownmiller, and Sun-Ok Lee

University of Arkansas

Objectives: The objectives of this study were to measure thephenolic contents of black and sumac grain sorghum bran and theirantioxidant capabilities and to determine the fermentation patternsof sorghum bran phenolic extracts in normal weight (NW) andoverweight/obese (O/O) individuals.

Methods:Black and sumac sorghumbran samples were analyzed fortotal phenolics (TP), total proanthocyanidins (TPC), total anthocyanins(ACY), and radical scavenging capabilities by the Folin-Ciocalteuassay, the 4-dimethylaminocinnamaldehyde (DMAC) assay, HPLC, andthe 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, respectively. Phenolicextracts were purified and dried to powder form. Fresh fecal sampleswere collected from 12 NW and 12 O/O individuals, incubated for24 h with anaerobic fermentation media, and subjected to 6 treatments:no treatment (blank), 75 mg fructooligosaccharides (FOS), 47 mg blacksorghum bran extract (B), 26 mg sumac sorghum bran extract (S),FOS + B, and FOS + S. Aliquots were collected at 0, 6, 12, 18, and24 h for analysis. Short-chain fatty acid production was determined byquantifying butyrate (BA), propionate (PA), and acetate (AA) throughgas chromatography. All statistical analyses were performed by two-way

ANOVA with Tukey’s test post hoc. Significance level was determinedas P < 0.05.

Results: Sumac bran had significantly higher TP (42.2 ± 1.6 mg/g)and TPC (7.8 ± 0.5 mg/g) than black bran (28.0 ± 1.6 mg TP/g and0.4 ± 0.1 mg TPC/g), whereas black bran had significantly higherlevels of ACY (1.2 ± 0.003 mg/g) than sumac bran (0.14 ± 0.01mg/g) (P < 0.01). Black bran polyphenols showed significantly greaterscavenging abilities than sumac bran (P< 0.01). Analysis of 12 subject’sdata showed that FOS + S trended higher than FOS in total SCFA, BA,PA, and AA production for the first 12 h in the NW group, and in BAfor the first 18 h in the O/O group.

Conclusions: Black and sumac sorghum bran are excellent sourcesof polyphenols and show considerable radical scavenging capabilities invitro. Gut microbial fermentation patterns may vary in response to FOScombined with varying classes of polyphenols from sorghum bran.

Funding SourcesArkansas Grain Sorghum Promotion Board.

Ingestion of Cheese Partially Recovers Cholic Acid-InducedDisorders Accompanied by Modulation of Bile Acid Metabolism(P08-057)

DongGeun Lee,1 Yutaro Takahashi,2 Tomoko Shimoda,2 YoshieKamo,2 Shota Hori,2 Taketo Hanai,2 Hidehisa Shimizu,3 HarutoKumura,2 and Satoshi Ishizuka2

1Hokkaido University, Division of Applied Bioscience, Japan; 2

Hokkaido University Research Faculty of Agriculture, Japan; and3Shimane University Faculty of Life and Environmental Science, Japan

Objectives:Bile acid (BA) is synthesized from cholesterol in the liverand is closely associated with lipid metabolism. High-fat diet inducesseveral metabolic disorders, including increasing gut permeability andhepatic lipid accumulation. In our previous study, we found that acholic acid (CA)-supplemented diet induced some of the disordersfound in high-fat diet consumption. Cheese is a nutritious fermenteddairy product and develops its own unique characteristics duringripening. It is reported that ingestion of ripened cheese improves lipidmetabolism, including reducing hepatic lipid accumulation and plasmacholesterol. However, little is known about the metabolic influenceinduced by cheese ingestion on BA metabolism. Therefore, this studywas conducted to investigate whether cheese ingestion affects BAmetabolism and further prevents the CA-induced disorders.

Methods: Cheese was made from skimmed milk and ripened for3 mo in a controlled condition. Four species of lactic acid bacteriawere used as a starter culture. Rats (WKAH/Hkmslc, 3 wk old) weredivided into 3 groups after 10 d of acclimation and fed different dietsfor 2 wk: control, CA-supplemented diet (CA), and CA with cheese-supplemented diet (CA-Ch). BAs were analyzed in the liver, portalblood, and fecal excretion at a molecular level by LC-MS. Triglyceride(TG) and cholesterol were analyzed in the liver, plasma, and feces. Inaddition, several parameters influencing CA-induced disorders weremeasured.

Results: After 2 wk of feeding, several CA-induced disorders wereobserved. Fecal CA excretions were ∼9.6 times higher in CA-fedrats than in CA-Ch-fed rats. The CA supplementation increased BAexcretion in the CA-fed rats. On the other hand, the CA-induced


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disorders were partially recovered by the cheese supplementation. It isconsidered that the cheese ingestion contributes to reduce fecal CA ex-cretion, which results in a reduction of DCA production and improvessymptoms of the CA-induced disorders. Calcium supplemented by theCA-Ch diet might reduce free BA as a BA sequestrant.

Conclusions: Cheese is a potent modulator of BA metabolism,and may serve as a nutraceutical to prevent some types of metabolicdisorders.

Chrysin Blocks Advanced Glycation End Product–AssociatedGlomerulosclerosis by Suppressing Autophagy in Mesangial Cellsand db/db Mice (P08-058)

Eun-Jung Lee and Young-Hee Kang


Objectives:Diabetic nephropathy (DN) is one of the major diabeticcomplications and the leading cause of end-stage renal disease.Advanced glycation end products (AGEs) play a causative role in thedevelopment of DN via induction of extracellular matrix accumulation.Recent research has demonstrated that autophagy is involved inthe development of DN. The aim of this study was to explore theinvolvement of autophagy and the mechanistic actions of chrysin inAGE-exposed mesangial fibrogenesis.

Methods: The in vitro study incubated human mesangial cellsexposed to 33 mM glucose and 100 μg/mL AGEs-BSA for 3 d inthe absence and presence of 1–20 μM chrysin (5,7-dihydroxyflavone)present in bee propolis and herbs. The in vivo study employed type 2diabetic db/db mice orally administered 10 mg/kg chrysin for 10 wk.

Results: Chrysin attenuated the formation of the stress fiber F-actinin 33 mM glucose– or AGE-BSA– exposed mesangial cells. Chrysinsuppressed the induction of microtubule-associated proteins 1A/1Blight chain 3B (LC3), beclin-1, and the autophagocytosis-associatedproteins Atg3 and Atg7 enhanced by AGE exposure in mesangialcells. In addition, chrysin inhibited the mesangial induction of thephosphorylated mechanistic target of rapamycin (p-mTOR) by AGEs.Oral supplementation of 10 mg/kg chrysin hindered the induction ofp-mTOR in db/db mouse glomeruli. Moreover, treating db/db micewith chrysin diminished the autophagy that was enhanced in diabeticglomeruli.

Conclusions: These results demonstrate that chrysin attenuatedAGE-induced mesangial fibrogenesis via the mTOR signal-mediatedinhibition of autophagy. These findings suggest the possibility thatchrysin could be a potential agent for the treatment of diabeticglomerulosclerosis.

Leucrose Improves Metabolic Abnormalities in High-Fat-Diet–Induced Obese Mice (P08-059)

Jihye Lee,1 Daeyeon Lee,2 Eunju Kim,1 Yuri Kim,1 and Sang-HoYoo2

1Ewha Womans University, South Korea; and 2Sejong University,South Korea

Objective: Obesity and metabolic dysregulation have dramaticallyincreased worldwide. High sugar intake is one of risk factors forobesity and its related metabolic abnormalities. In the present study, we

investigated the potential effect of leucrose, a natural sucrose isomer, onblood glucose and hepatic lipid in high-fat-diet (HFD)–induced obesemice.

Methods: Male C57BL/6 mice were fed AIN-3G diet (Ctrl), 60%HFD (OB), or HFD, where total sucrose content in the diet wassubstituted with leucrose at 25% (L25) or 50% (L50) for 12 wk.Fasting blood glucose and blood lipid profiles were determined.Histologic changes, triglyceride (TG) content, and mRNA levels ofadipogenesis and β-oxidation–related genes in liver tissues wereanalyzed.

Results: Leucrose supplementation did not affect body weight andfat mass. However, the relative liver weight was significantly loweredin the L50 group compared with the OB group, and fasting bloodglucose levels were decreased in the L25 and L50 groups comparedwith the OB group. In addition, leucrose significantly lowered hepaticTG content in the L50 group. mRNA levels of lipid metabolism–related genes, including peroxisome proliferator-activated receptorgamma PPARγ and sterol regulatory element-binding protein 1-c(SREBP-1C), were downregulated, whereas lipid β-oxidation–relatedgenes, such as carnitine palmitoyltransferase 1A (CPT1A) and acetyl-CoA oxidase (ACO), in the liver tissue were upregulated by leucrosesupplementation.

Conclusions: These result suggested that leucrose may improveobesity-related metabolic dysregulation by inhibiting blood glucoseincrease and hepatic lipid accumulation.

Funding SourcesThis research was supported by National Research Foundation of

Korea (NRF-2015R1A2A1A15056119).

Anti-Obesity Effects of Dietary Garlic Powder in PeroxisomeProliferator-Activated γ Receptor Coactivator-1 α (PGC-1α)-Luciferase Transgenic Mice (P08-060)

Mak-Soon Lee, Seohyun Lee, Soojin Lee, and Yangha Kim

Ewha Womans University, South Korea

Objective: The peroxisome-proliferator–activated receptor γ

coactivator-1α (PGC-1α) plays an important role in the maintenanceof lipid, glucose, and energy homeostasis, and thus is related tometabolic diseases such as obesity and diabetes. This study wasperformed to investigate the antiobesity effect of garlic powder intransgenic (TG) mice of PGC-1α-luciferase.

Methods: To examine the control function of PGC-1α, we bredthe TG mice with a PGC-1α promoter (−970/+412 bp) containingluciferase as a reporter gene. PGC-1α-luciferase TG mice were fed a45%high-fat diet for 8wk to induce obesity. Subsequently, themiceweremaintained on either a high-fat control diet (TG-CON), or high-fat dietssupplemented with 2% (TG-GP2) or 5% (TG-GP5) garlic powder for afurther 8 wk.

Results: Dietary garlic powder reduced body weight in the TG-GL2 and TG-GL5 groups compared with the TG-CON group. Fur-thermore, garlic supplementation significantly decreased plasma levelsof triglycerides, total cholesterol, and leptin in the TG-GL5 groupscompared with the TG-CON group. Specifically, luciferase activity inmetabolic tissues such as liver, white adipose tissue (WAT), and brown


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adipose tissue (BAT) was increased by garlic supplementation in a dose-dependent manner.

Conclusions: These results suggest that the antiobesity effect ofgarlic powder was associated with the increase of PGC-1α luciferaseactivity in liver, WAT, and BAT of PGC-1α-luciferase TG mice.Therefore, this TG system may be useful in evaluating the antiobesityefficacy of various health functional foods.

Hypocholesterolemic Effect of Nanoemulsion Quercetin in RatsFed High-Cholesterol Diet (P08-061)

Soojin Lee,1 Hye-yeon Son,1 Mak-soon Lee,1 Eugene Chang,1Seog-Young Kim,1 Hyunmi Ko,1 Chong-Tai kim,2 and YanghaKim1

1EwhaWomans University, South Korea; and 2Korea Food ResearchInstitute, South Korea

Objective: This study aimed at investigating the hypocholes-terolemic effect of quercetin and nanoemulsion quercetin in rats fed ahigh-cholesterol diet.

Methods: Male Sprague-Dawley rats (6 wk old) were randomlydivided into 6 groups as follows: a normal diet (NOR), high-cholesteroldiet (HC), and HC containing 0.05% quercetin (LQ), 0.1% quercetin(HQ), 0.05% nanoemulsion quercetin (LNQ), or 0.1% nanoemulsionquercetin (HNQ). All groups of rats were fed each experimental diet for4 wk.

Results: At the end of the experiment, serum levels of totalcholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) werelower in the HNQ group than in the HC group (P < 0.05). Thehepatic level of TC was lower in the LNQ group than in the HC group(P < 0.05). The fecal excretion of triglycerides (TG), TC and total bileacids (TBA) was higher in the nanoemulsion quercetin–supplementedgroups than in the HC group (P < 0.05), whereas quercetin did notshow this effect. In addition, the mRNA expression of genes involvedin hepatic bile acid synthesis and cholesterol efflux, such as LiverX receptor α (LXRα), ATP binding cassette subfamily G member 5(ABCG5), ATP binding cassette subfamily G member 8 (ABCG8), andcholesterol 7α-hydroxylase (CYP7A1), was upregulated in the liver ofrats supplementedwith nanoemulsion quercetin comparedwith theHCgroup (P < 0.05), whereas quercetin did not show this effect. Further,the activity of CYP7A1 in liver was enhanced by the nanoemulsionquercetin supplementation (P < 0.05), but not by quercetin.

Conclusions: These results suggest that nanoemulsion quercetinwould have beneficial effects on hypercholesterolemia in vivo, possiblythrough synthesis of hepatic bile acid and excretion of cholesterol.

Funding SourcesThis study was supported by a grant from the Ottogi Foundation in

Korea.

Colonic Metabolites of Resveratrol are Key for Anti-Inflammation (P08-062)

Fang Li, Yanhui Han, Qi Wang, Zili Gao, Zhengze Li, MinqiWang, and Hang Xiao

University of Massachusetts-Amherst

Objective: Resveratrol has been reported to exert various health-promoting activities, including protective effects against colonic in-flammation. However, the oral bioavailability of resveratrol is very low.In order to understand the mechanism underlying this paradox (lowbioavailability but high bioactivity) of resveratrol, we identified andquantified the major metabolites of resveratrol in mouse colon anddetermined their anti-inflammatory effects in comparison with thoseof resveratrol.

Methods: Major colonic metabolites were identified and quanti-fied with HPLC-MS. To understand the role of colonic metabolitesof resveratrol in anti-inflammation, the inhibitory effects of themetabolites, resveratrol, and their combinations at the concentrationsequivalent to those found in the mouse colonic tissues were deter-mined in lipopolysaccharide-induced inflammation in both RAW264.7macrophages and a cell line with TLR-4–specific inflammation.

Results: Our results showed that the major metabolites found inthe colonic mucosa of the mice fed physiologically relevant amountof resveratrol were dehydroresveratrol and lunularin. The tissue levelsof these metabolites were ∼25-fold higher than that of resveratrol inthe mouse colonic mucosa. The cell culture results demonstrated thatthe anti-inflammatory effects of the colonic metabolites were muchstronger than that of resveratrol at the relevant tissue levels; moreover,the combination of the colonicmetabolites and resveratrol produced thestrongest anti-inflammatory effects and the contribution of resveratrolwasmarginal. It was also found that the anti-inflammatory effects of themetabolites were specific to TLR-4–mediated pathways.

Conclusions: Overall, this study revealed that dehydroresveratroland lunularin were the major colonic metabolites of resveratrol, andthey potentially play much more important roles in anti-inflammationin the colon than resveratrol.

Funding SourcesUSDA.

Complexation with Phenolic Acids Affects both RheologicalProperties andDigestibility ofMaizeAmylopectin andPotato Starch(P08-063)

Min Li,1 Cheikh Ndiave,2 E. Allen Foegeding,1 Chris Pernell,1and Mario G. Ferruzzi1

1North Carolina State University; and 2Purdue University, IN

Objective: The aim of this study was to characterize the structural,functional, and digestibility changes in maize amylopectin (MA) andpotato starch (PS) resulting from complexations with phenolics.

Methods: Caffeic acid, ferulic acid, or gallic acid were complexedwith MA and PS, respectively. Phenolic contents of native starch andstarch complexes were determined by LC-MS. Rheological propertieswere determined by rapid viscometry analysis and differential scanningcalorimetry. Rapidly digestible starch, slowly digestible starch (SDS),and resistant starch (RS) contents were measured in the presence andabsence of phenolic complexations. In addition, Fourier transforminfrared (FTIR) and nuclear magnetic resonance (NMR) spectrometry,and iodine-binding techniques were used to explore the molecularmechanisms underlying the changes in rheology and digestibility.

Results: Complexation increased the phenolic content of bothnative starches. Complexed phenolics decreased the thermal stability


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of native MA (70.6 ± 0.18°C) and PS (64.7 ± 0.14°C) by reducingmelting temperatures to 69.2–69.4°C and 58.6–58.8°C, respectively.Significantly reductions of swelling factors were observed for both MA(30.4 ± 3.1 versus 21.4–25.1) and PS (40.9 ± 4.2 versus 22.0–26.1;P < 0.05). Peak viscosity, hot paste viscosity, and cool paste viscosityof starch-phenolic complexes were 2–30 times lower than those ofnative starches and uncomplexed starch-phenolic mixtures. More in-terestingly, complexed phenolics appeared to have additional inhibitoryeffects on starch digestion as evidenced by the modest increase in SDScontents for MA and RS contents for PS, when compared with thematching starch-phenolic mixtures. These alterations were tentativelyattributed to disruptions of starch crystallinity and α (1,4) glycosidiclinkage, as reductions in short-range orders of starch granules and 1H(1,4) signals were discovered by FTIR and NMR, respectively.

Conclusions: Complexation of phenolics could serve as an impor-tant factor in modulating the rheologic properties and digestibility ofstarch. Although complexation appears to lead to structural alterationsthat thermally destabilize starch granules and reduces viscosity ofgelatinized MA and PS, these changes may modestly delay starchdigestion in vivo.

Funding SourcesThis study was supported by in part by a grant from the Alliance for

Potato Research and Education and NC Agricultural Research Service.

Potato Product Form Affects In Vitro Starch Digestibility andGlucose Transport but only Modestly Impacts 24-h Blood GlucoseResponse in Humans (P08-064)

Min Li,1 Judy George,2 Stephanie Hunter,2 Bruce Hamaker,2Richard Mattes,2 and Mario Ferruzzi1

1North Carolina State University; and 2Purdue University, IN

Objectives: White potatoes are a rich source of dietary phenolicsknown to interact with starch digestion. However, little is knownregarding the relationship between these factors and glycemic responsefrom consumer products. This study assessed the glycemic impactsof white potato phenolics and whether consumption of potatoes atbreakfast would lead to a reduction in 24-h glycemic impact andsubsequent appetite.

Methods: White potatoes (Shepody, SH; Russet Burbank, RB)were obtained fresh and as commercial frozen products (French Fry,Home Fry, Hash Brown). Phenolic content was measured by LC-MS.Total (TS), slowly digestible (SDS), and resistant starch (RS) contentswere determined enzymatically. Starch digestion and intestinal glucosetransport were screened with an in vitro digestion/Caco-2 humanintestinal cell model. The impact of phenolic-rich potatoes on 24-hglycemic response and subsequent appetite was assessedwith 24 healthyadults (12 male, 12 female) randomly assigned in a crossover design.Participants consumed 1 of 4 serving (50 g available carbs) of RBfrozen products or a wheat pancake (control) at breakfast followed bya standardized lunch and released. Glucose response was monitoredthrough the day by continuous glucosemonitoring. Appetitive behaviorwas determined as well by visual analog scales.

Results: The phenolic content of commercial RB French frieswas higher (217 mg/100 g dw) relative to RB and SH home friesand hash browns (56–67 mg/100 g dw). The RS:TS ratio was higher

in frozen products (5.4–18.3%) compared with freshly prepared(3.6–7.6%). In vitro glucose release (31.8–36.9 mM) was similaracross products, whereas intestinal glucose transport was significantlylower (P

Conclusions: Potato processing, product form, and phenolic con-tent might affect RS formation, but subsequent effects on glycemicprofiles remain limited to acute exposure and may not extendthroughout the day.

Funding SourcesGrant provided by the Alliance for Potato Research and Education.

Effects of 2 Weeks of Daily Mango Fruit Intake on VascularFunction, Blood Pressure, and Gut Fermentation in Healthy AdultWomen (P08-065)

Xiang Li,1 Matthew Vanness,1 Roberta R. Holt,1 William F.Horn,2 Nancy L. Keim,2 Carl Keen,1 JohnG. Carson,1 andRobertM. Hackman1

1University of California, Davis; and 2Western Human NutritionResearch Center

Objectives: Mangos are rich in mangiferin, a phenolic acid thathas multiple bioactive effects. Combined with carotenoids, fiber, andother nutrients, mangos may be of benefit to vascular health. Thisstudy assessed whether a short-term (14 d) or acute (2 h) intakeof mangos can influence the following properties: 1) microvascularfunction and the augmentation index, determined by peripheral arterialtonometry; 2) blood pressure; 3) optical platelet aggregometry, and4) gut fermentation, determined by breath hydrogen and methane, inhealthy adult women.

Methods: Twenty-five healthy postmenopausal females (body massindex 25–40 kg/m2) were assessed at 3 study visits. Study visit 1 (SV1)started a run-in period of 14 d duringwhich nomangoswere consumed,with baseline and 2 h measures taken. At study visit 2 (SV2), baseline(0 h) measures were taken, followed by ingestion of 300 g (2 cups) offresh, frozenmangos, and data were collected 2 h later. Participants thenconsumed 300 g of mangos daily for 14 d, followed by assessment atstudy visit 3 (SV3), which followed the same protocol as SV2. Breathsamples were collected at baseline at each study visit.

Results: A significant interaction between treatment and time(P= 0.005) was observed for systolic blood pressure (SBP). At baseline,SBP was not significantly different between study visits; however, SBPwas significantly lower 2 h after mango intake during SV2 and SV3compared with no mango intake during SV1 (112 ± 9 mm Hg SV2compared with 116 ± 12 mm Hg SV1, P = 0.013; 111 ± 11 mm HgSV3 compared with 116 ± 12 mm Hg SV1, P = 0.003). A significanttreatment effect was noted formean arterial pressure withmango intakecompared with no mango intake (87 ± 7 mm Hg SV2 compared with89 ± 9 mm Hg SV1, P = 0.04; 86 ± 8 mm Hg SV3 compared with89± 9mmHg SV1,P= 0.005). Pulse pressurewas significantly reduced2 h post-intake compared with baseline during SV2 (41 ± 7 mm Hgbaseline compared with 38 ± 5 mm Hg at 2 h, P = 0.005). Breathmethane was significantly reduced in 3 of 6 participants that producedmethane.

Conclusions: Two cups of mango intake had acute (2 h) beneficialeffects on blood pressure in healthy postmenopausal women. A number


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of women showed favorable changes in breath methane, an indicationof the potential influence of mango intake on gut fermentation.

Funding SourcesSupported in part by funds from the National Mango Board,

Orlando, FL and USDA CRIS 2032 51530 02200D.

Identification of Colonic Metabolites of Curcumin and theirSynergistic Interaction with Curcumin in Anti-Inflammation (P08-066)

Zhengze Li,1 Che Pan,1 Mingyue Song,2 Haiyan Luo,1 and HangXiao1

1University of Massachusetts Amherst; 2South China AgriculturalUniversity, China

Objective: It is well known that dietary intake of curcumin canproduce anti-inflammatory effects in the colon. However, themetabolicfate of curcumin in the colon and the role of the colonic metabolitesof curcumin in anti-inflammation remain unknown. The aim of thiscell culture study was to identify the colonic metabolites of curcuminin mice fed curcumin and to determine the role of these metabolites inanti-inflammation.

Methods: After oral administration of curcumin for 6 wk, 4major curcumin metabolites were identified in the colonic mucosaof the mice: tetrahydrocurcumin (THC), hexahydrocurcumin (HHC),octahydrocurcumin (OHC), and ferulic acid (FA). More importantly,the abundance of these metabolites was similar or even higher thanthat of curcumin in the colonic mucosa. The anti-inflammatory effectsof curcumin, its metabolites, and their combinations were determinedin lipopolysaccharide (LPS)-treated RAW264.7 macrophages at theconcentrations equivalent to those found in the colonic mucosa of thecurcumin-fed mice.

Results: The results showed that curcumin showed a dose-dependent inhibition on the production of nitric oxide (NO) inducedby LPS in the macrophages, whereas the mixture of 4 metabolites didnot significantly inhibit NO production. However, the combinationof curcumin and the metabolites showed much stronger inhibitoryeffects than that of curcumin or the metabolite mixture alone. Moreimportantly, the isobologram analysis confirmed that the enhancedanti-inflammatory effects of the combination of curcumin and themetabolites were highly synergistic. The Western blotting analysisdemonstrated that the combination of curcumin and its metaboliteseffectively suppressed the expression of iNOS andCOX-2 in LPS-treatedmacrophages, and these effects were stronger than that produced bycurcumin or the metabolite mixture alone.

Conclusions: Our findings demonstrated the significant role of thecolonic metabolites of curcumin in mediating the anti-inflammatoryeffects of orally administered curcumin, and suggest a novelmechanismfor the anti-inflammatory effects of curcumin.

Funding SourcesThis study was partially supported by funds from the USDA.

Effects of Glutamine and n–3 Polyunsaturated Fatty Acid–Supplemented Resuscitation Fluid on Oxidative Stress and Inflam-matory Mediators in Trauma-Hemorrhagic Shock (P08-067)

Bing Xiang Liu,1 Lo Hui-Chen,1 Lee Chien Hsing,2 and Wu YiChen,1

1Nutritional Science, Fu Jen Catholic University, New TaipeiCity, Taiwan; and 2Division of Pediatric Surgery, Department ofSurgery, Children’s Hospital of China Medical University, Taichung,Taiwan

Objective:Trauma-hemorrhagic shock and reperfusion (THR) is anemergent condition accompanied by a cascade production of reactiveoxygen species (ROS) and inflammatory mediators. Glutamine, aprecursor of de novo antioxidant glutathione, has been shown torestore hepatic ATP depletion and reduce hepatic damage in THRrats. In addition, n–3 (ω-3) polyunsaturated fatty acids derived fromfish oil are precursors of pro-resolving mediators in inflammationresolution. Therefore, the aim of this study is to investigate theeffects of glutamine– and fish oil–supplemented resuscitation fluidson oxidative stress and inflammatory mediators of the liver and lungin THR.

Methods: THR rats were given a 5-cm midline laparotomy andcatheterizations in the left carotid artery and right jugular vein. Blooddrawn from the left carotid artery reached a mean arterial pressure of30–35 mm Hg within 10 min and maintained hypovolemia for 60 min;the rats were subsequently resuscitated with shed blood and an equalvolume of lactate Ringer’s solution with or without both l-alanyl-l-glutamine and fish oil within 10 min, followed by a continuous, lowinfusion rate of resuscitation fluids. Healthy and sham-operated ratswere included as controls.

Results:After 42 h of infusion, THR rats had significantly increasedrelative lung weights and decreased liver glutathione peroxidase(GPx) activity (one-way ANOVA, P < 0.05) compared with thecontrols. The THR-increased hepatic TBARS, i.e., the index of lipidperoxidation, were decreased by glutamine and fish oil and theincreased pulmonary TBARS were decreased by glutamine (two-wayANOVA, P < 0.05). In THR rats, glutamine was the main factorincreasing hepatic interferon-γ and interleukin (IL)-6 and pulmonaryintercellular adhesion molecule (ICAM)-1, non-protein thiol group,and GPX activity. Hepatic IL-6 and ICAM-1 and pulmonary IL-4 and ICAM-1 were significantly decreased by fish oil, and theseeffects were attenuated when combined with glutamine. Moreover, acombination treatment of glutamine and fish oil significantly decreasedpulmonary IL-8.

Conclusion:These results suggest that both glutamine– and fish oil–supplemented resuscitation fluids may decrease antioxidative capacity,and fish oil–supplemented resuscitation fluids may further decreaseinflammatory mediators in the liver in THR.

Funding SourcesNSC 102-2320-B-030-005-MY3.


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Effects of ageLOC Defense Formula on Immune Regulation andDetoxification: Phenotype and Molecular Evaluation (P08-068)

Jihong Lu,1 Yiping Ren,1 Doug Stevenson,2 and Mark Bartlett2

1Nu Skin (China) Daily-Use & Health Products Co. Ltd, Centerof Anti-Aging Research, Shanghai, China; and 2Nu Skin Enterprises,Center of Anti-Aging Research, Provo, UT

Objective: Through the use of a high-throughput gene expressionand epigenetics–based product screening platform and phenotypesubstantiation, we found several ingredients to have cellular protectionand antiaging characteristics. We have created an antiaging formula,ageLOC defense formula (ADF), that includes Ganoderma lucidum(Reishi), broccoli seed, and Maitake extracts. An aged mice model wasused to evaluate cellular protection effects of this formula.

Methods:Mice aged 18 mo were randomly assigned to aged control(AC, n= 17) and ADF (n= 16) groups. Mice aged 16 wk were includedas young controls (YC, n = 17). ADF was fed at 264 mg/kg bodyweight to the mice for 8 wk in the ADF group. Biomarkers for immuneregulation, inflammation, and detoxificationwere detected in blood andliver. Gene expressions in liver and lung were also detected.

Results: Nrf2 protein was significantly deceased (P < 0.05) in liverin theADF andYC groups comparedwith theAC (Figure 1). Significant

FIGURE P08-068-1 Liver Nrf2 protein is significantly decreased in young controls and ageLOC defense-fed mice compared with agedcontrols.

FIGURE P08-068-2 Significant increases in IL-13 in young controls and ageLOC defense-fed mice compared with aged controls.

increases in interleukin (IL)-13 were observed in both the ADF andYC groups (P < 0.05) (Figure 2). Gene expression data were analyzedby KEGG and IPA. The ADF and AC groups, and the AC and YCgroups were compared. In liver, reversed regulation of PPAR signalingand arachidonic acid metabolism pathways was found in these twocomparisons. ADF consumption activated the lipopolysaccharide/IL-1–mediated inhibition of the RXR function pathway in the ACcompared with the YC group; the upstream regulator interferon (INF)γ was activated, and INFaR1 and PPARa were inactivated, in theADF compared with the AC group (Figure 3). In lung, cytokine-cytokine receptor interaction, arachidonic acidmetabolism, and asthmapathways were reversely regulated in the ADF compared with theAC group, and in the AC compared with the YC group (Figure 4).The upstream regulators PPARg and IL-10 were inactivated in theADF compared with the AC group. Analysis of the effects of theseregulators revealed that the inactivation of IL-10 may cause activationof inflammation responses, movement of blood cells, and chemotaxis(Figure 5).

Conclusions: The results indicate that 8 wk of consumption ofageLOC defense formula by aged mice may regulate the immuneresponse and detoxification via Nrf2 protein and TH2 cytokine IL-13 phenotypically, and regulate the PPARγ signaling pathway at themolecular level.


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FIGURE P08-068-3 Regulation of upstream regulators in liver after ageLOC defense consumption.

FIGURE P08-068-4 Regulation of upstream regulators in lung after ageLOC defense consumption.


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FIGURE P08-068-5 Analysis of the effects of regulators in lung after ageLOC defense consumption.

Activation of the Novel Type 2 Diabetes Markers Free Fatty AcidReceptor-1 andGlucokinase byAnthocyanins fromColoredCorn InVitro (P08-069)

Diego Luna-Vital and Elvira Gonzalez de Mejia

University of Illinois at Urbana-Champaign

Objective: The aim of this study was to evaluate the ability ofanthocyanins in colored corn to interact and activate the free fattyacid receptor-1 (FFAR1) and glucokinase (GK) in pancreatic cells andhepatocytes, respectively. Our hypothesis was that pure anthocyanins,and anthocyanin-rich extracts from colored corn, would interact withFFAR1 and GK to increase insulin secretion in INS-1E pancreatic cells,and glucose uptake in HepG2 hepatic cells.

Methods: Through the use of a dual-layer cell culture with Caco-2 cells plus INS-1E or Caco-2 cells plus HepG2, cells were treatedwith an anthocyanin-rich extract from the pericarp of purple corn(PCW) and red corn (RCW), as well as the pure anthocyanins cyanidin-3-O-glucoside (C3G), peonidin-3-O-glucoside, and pelargonidin-3-O-glucoside. In addition, the semipurified C3G (C3Gp) and condensedforms (CFp) isolated from purple corn were used in the study.An indirect biochemical assay quantified the intermediate secondmessenger inositol monophosphate, and through ELISA. Glucoseuptake was determined by remnant glucose quantification. Througha biochemical assay, the formation of NADPH determined activationof GK.

Results: At a concentration of 100 µM, the pure and semipurifiedanthocyanins enhanced the glucose-stimulated insulin secretion (GSIS)in INS-1E cells ranging from 18% to 40% higher than untreated cells(P < 0.05). PCW and RCW increased the GSIS by 51% and 40%,respectively. When the anthocyanins and extracts were combined withthe FFAR1-specific inhibitor GW-1100, their efficacy was reduced byup to 20%, suggesting that FFAR1 activation is playing a role in theenhanced GSIS. It was determined that C3G was the most effectiveanthocyanin activating FFAR1 (EC50: 245.4 µM). Both PCW and RCW

had a comparable activating potential on FFAR1 (EC50: 77 µg/mL).Regarding hepatic HepG2 cells, the treatment with 100 µM of P3G,C3Gp and CFp increased (P < 0.05) the glucose uptake by 19%, 31%,and 18%, respectively. PCW and RCW increased the glucose uptake inHepG2 cells by 48% and 37%, respectively. It was determined that CF-Pwas the most effective anthocyanin activating GK activity (EC50:39.9 µM) and the extracts had similar efficacy (EC50:44 µg/mL).

Conclusion:Our results suggest that anthocyanins frompurple corninteract with novel biomarkers FFAR1 and GK to ameliorate type 2diabetes comorbidities.

Funding SourcesUSDA National Institute of Food and Agriculture Hatch 1014457.

Improved Effects of Sea Cucumber Collagen Oligopeptides onLiver Injury Induced by Antituberculosis Drugs (P08-070)

Aiguo Ma,1 Jing Cai,2 Ya Chen,2 and Yong Li3

1Institute of Nutrition and Health and 2College of Public Health,Qingdao University; and 3College of Public Health, Peking University,China

Objective: The use of antituberculosis drugs can cause manyside effects, specifically liver injury. This study aimed to explore theprotective effect of sea cucumber collagen oligopeptides (SCCOPs) onliver injury induced by the combination of rifampin and isoniazid inrats.

Methods: Seventy-two male Wistar rats were randomly dividedinto 6 groups: blank control group (A), model group (B), positivecontrol group (C), low-dose (D), mid-dose (E), and high-dose (F)SCCOP groups (each n = 12). Group A rats were given 0.9% saline(20 mg/kg body wt) by gavage, and isoniazid (50 mg/kg body wt)and rifampicin (50 mg/kg body wt) were given by gavage once aday in the other groups. After 2 h, silybin was given (22.1 mg/kg


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body wt) by gavage in group C, and groups D, E, and F weregiven SCCOPs by gavage (346, 692, and 1038 mg/kg body wt,respectively). The whole experiment continued for 4 wk. Hematoxylinand eosin (HE) staining was used to observe the histomorphology andhistopathologic grading of liver tissue. Serum alanine aminotransferase(ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP),liver index, interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α and the oxidative stress indexes of liver homogenate were alltested.

Results: There were injuries in liver cells by HE staining and liverindex in group B, such as swelling, unclear boundaries, fatty degen-eration, inflammatory cell infiltration, etc. The liver histomorphologywas significantly improved in groups C, D, E, and F. Compared withgroup A, ALT and ALP were increased by 6.46% and 17.70% in groupB (P< 0.05). Compared with group B, ALT and ALP were decreased by25.36% and 29.65% in group C (P< 0.05), and with the use of SCCOPs,ALT and ALP were all decreased by 28.89%, 27.77%, and 18.56% in theformer, and by 14.84%, 24.78%m and 21.08% in the latter in groups D,E, and F, respectively (P < 0.05). The levels of IL-6 and TNF-α weresignificantly lower in groups C, D, E, and F from liver homogenate thanthese of group B, whereas IL-10 was much lower than these groups(P < 0.05).

Conclusions: To some degree, SCCOPs can improve liver injuryinduced by antituberculosis drugs.

Funding SourcesNational Natural Science Funds (81673160).

Extract from Hawthorn Fruit Induces Cell Cycle Arrest andApoptosis in Human Colon Cancer Cells (P08-071)

LeiMa,GuanyingXu, XinyuTang,MingzhuCai, andHongChen

University of Illinois at Urbana-Champaign

FIGURE P08-071-1 Immunofluorescence staining.

Objective: Crataegus pinnatifida, commonly named hawthorn, hasbeen used in China and other Asian countries for >2000 y, both as atraditional medicinal herb and as a food component. The objective ofthe study was to investigate the anticancer activities of extract from C.pinnatifida (ECP) and related molecular mechanisms.

Methods: The ground hawthorn was subjected to distilled waterextraction, followed by 70–85% ethanol precipitation, and lyophiliza-tion. Human colon cancer cells (HCT116) were treated with ECP atconcentrations of 125, 250, 500, 1000 μg/mL for 12 h. Phospho-AKTand Ki-67 expression were assessed by immunofluorescence staining.Cell cycle and apoptosis were analyzed by flow cytometry. mRNAexpression was examined by real-time RT-PCR.

Results: Ki-67 protein staining was reduced by ECP treatment incells, and phospho-AKT protein expression was significantly reducedwith the ECP treatments. The proportion of apoptotic cells wassignificantly increased by ECP. Concomitantly, ECP treatment inducedG1 phase arrest in HCT116 cells. HCT116 cells treated with 500 and1000 μg/mL ECP had significantly increased expression of caspase 3,caspase 7, caspase 8, and caspase 9. ECP also upregulated the mRNAlevel of FADD, TRADD, and TNFR1 in a dose-dependent manner. ECPdownregulated the CyclinD1 mRNA expression.

Conclusions: Our findings demonstrated that ECP exhibited anti-cancer activities in HCT116 cells by induction of the apoptosis pathway.ECP treatment also decreased proliferation by induction of G1 phasecell cycle arrest. The results indicate somepotential benefits of providinghawthorn extracts as functional foods for cancer patients. Furthermechanistic studies are warranted to establish the molecular pathwaysthat are involved in the anticancer effects of ECP.

Funding SourcesUSDA Cooperative State Research, Education and Extension Ser-

vice, Hatch projects ILLU-698-394 and ILLU-698-379.



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FIGURE P08-071-2 Cell apoptosis.


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FIGURE P08-071-3 Cell cycle.

Identification of the Anti-Inflammatory Ingredients from Cir-sium japonicum and the Anti-inflammatoryMechanism of Luteolin-7-O-β-d-Glucuronide (P08-072)

Qin Ma and Jianguo Jiang

South China University of Technology, China

Objective: Cirsium japonicum is listed as a health food in China,and is used for cooling blood and achieving hemostasis. The aim ofthis study was to identify its active components with anti-inflammationactivity.

Methods: C. japonicum was extract with 70% ethanol, and thenloaded onto a macroporous resin column chromatography to obtain 5fractions. These fractions were then separated through the use of silicagel column chromatography. To evaluate the anti-inflammation activity,lipopolysaccharide-induced RAW 264.7 cells were used to measure NOproduction, inflammation-related mRNA, and protein expression.

Results: The fraction CJ-50 exhibited the best anti-inflammationeffect. Six compounds, namely luteolin-7-O-β-d-glucuronide (LG),luteolin-7-O-β-d-glucuronide methyl ester, apigenin-7-O-β-d-glucuronide (AG), linarin, apigenin-7-O-β-d-glucuronide methylester, and pectolinarin, were isolated and identified from the fraction.The 6 flavonoid compounds exhibited different degrees of anti-inflammatory activity; LG and AG showed stronger activity thanthe others. Further research showed that LG can inhibit NO andproinflammatory cytokine production, likely resulting from inhibitingthe phosphorylation of JNK.

Conclusions: LG may be a therapeutic candidate for the anti-inflammation activity of C. japonicum as well as a dietary complementfor health promotion.

Funding SourcesScience and Technology Project of Guangzhou City

(201604020150).



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FIGURE P08-072-1 Effect of isolated compounds on cell viability and lipopolysaccharide-induced NO production in RAW 264.7 cells.

FIGURE P08-072-2 Effect of luteolin-7-O-β-D-glucuronide on lipopolysaccharide-induced proinflammatory cytokine production.


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FIGURE P08-072-3 Effect of luteolin-7-O-β-D-glucuronide on IL-6, TNF-α, COX-2, and iNOS mRNA expression.

FIGURE P08-072-4 Effect of luteolin-7-O-β-D-glucuronide on COX-2 and p-JNK expression.


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MuscadineGrapeExtract Inhibits Proliferation ofHER2-PositiveBreast Cancer Cells in Association with Reduced AKT Signaling(P08-073)

Jessica Mackert, Elisabeth Tallant, and Patricia Gallagher


Objective: Targeted drugs, such as trastuzumab, are effective forsome patients with human epidermal growth factor receptor 2 (HER2)–positive breast cancer. Unfortunately, 20% of early-stage patients and70%of patientswithmetastasis do not respond, and 70%of patientswhoinitially respond progress to metastatic disease, highlighting the needfor additional therapies. We determined whether a muscadine grapeskin and seed extract (MGE) inhibits the proliferation of HER2-positivebreast cancer cells.

Methods:HumanHER2-positive breast cancer cells were incubatedwith MGE and proliferation was analyzed with an IncuCyte ZOOMsystem. Protein expression was determined by Western blot.

Results: Increasing concentrations of MGE (10–40 μg/mL) sig-nificantly inhibited proliferation of trastuzumab-sensitive SKBR3 andtrastuzumab-resistant HCC1954 human HER2-overexpressing breastcancer cells in a time- and dose-dependent manner. MGE (20–30μg/mL, 24 h) significantly reduced phospho-AKT (Ser473) by 63% inSKBR3 (n = 3) and by 50% in HCC1954 (n = 5) cells. Phosphorylatedphosphoinositide-dependent kinase-1 (PDK1), which activates proteinkinase B (AKT), was also significantly reduced by 23% with 24 hMGE treatment (20 μg/mL) in SKBR3 cells (n = 4). AKT activationinhibits Forkhead box protein (FOXO) transcription factor, decreasingexpression of negative cell-cycle regulators, including cyclin-dependentkinase inhibitor 1B (p27), resulting in increased proliferation. FOX01and p27 were increased 4-fold (n = 3) and 2-fold (n = 4), respectively,in SKBR3 cells followingMGE incubation, whereas the extract caused a12-fold increase in FOX01 (n = 3) and a 2-fold increase in p27 (n = 4)in trastuzumab-resistant HCC1954 cells. These results suggest thatMGE decreases PDK1 to reduce activated AKT, resulting in increasedFOX01 and p27 to inhibit cell proliferation. S-phase kinase-associatedprotein 2 (SKP2) which targets p27 and FOX01 for degradation was alsosignificantly reduced by 67% in SKBR3 (n= 4) and by 74% inHCC1954(n = 4) cells with 24 h MGE treatment.

Conclusions: MGE inhibits proliferation of trastuzumab-sensitiveand -resistant HER2-positive breast cancer cells through regulation ofAKT signaling, suggesting that it may serve as a medical food for thetreatment of HER2-positive breast cancer.


Effect of Supplementation with Pumpkin Seed Oil Comparedwith Pumpkin Seeds on Blood Pressure and Menopausal Symptomsin Normotensive Postmenopausal Women (P08-074)

Madhura Maiya and Carolyn Moore

Texas Woman’s University

Objective: This study compared the effect of supplementation withpumpkin seeds or pumpkin seed oil (PSO) for 12 wk on blood pressure(BP, systolic and diastolic), endothelial function, plasma lipids, C-reactive protein (CRP) concentrations, and menopausal symptoms innormotensive postmenopausal women.

Methods: In this randomized trial postmenopausal women (n= 27)were randomly assigned to receive raw pumpkin seeds (4.1 g/d)or PSO (2 g/d) for 12 wk. BP, plasma lipids, endothelial function(EndoPAT 2000, Itamar-medical), and CRP concentrations weremeasured at baseline and 12 wk following supplementation. Par-ticipants also completed a menopausal symptom questionnaire atbaseline and at 12 wk. Statistical analyses were performed with SPSSversion 24. Both within-subjects and between-subject effects werecompared by repeated-measures ANOVA. An α of 0.05 was set forsignificance.

Results: In the pumpkin seed group, systolic BP significantlydecreased from baseline (118 ± 12 to 114 ± 14 mm Hg; P = 0.014)compared with the PSO group (no significant changes). Diastolic BPat 12 wk decreased significantly from baseline in the PSO group(75 ± 9 to 72 ± 9 mm Hg; P = 0.026) and in the pumpkinseed group (74 ± 6 to 71 ± 8 mm Hg; P = 0.002). A trendingimprovement in endothelial function at 12 wk was reflected by anonsignificant increase in reactive hyperemia index (4%) and a decreasein augmentation index (25%) in the pumpkin seed group. Overall,menopausal symptom score significantly decreased for women in thePSO group from baseline (15.7 ± 7) to 12 wk (8.5 ± 6; P < 0.01) witha significant decrease in severity of hot flushes (P = 0.02). There wereno significant changes in plasma lipid or CRP concentrations in bothgroups.

Conclusion: Pumpkin seeds exhibited cardioprotective benefits bysignificantly reducing BP and by providing a marginal improvementof endothelial function in postmenopausal women. Further studiesat different doses of pumpkin seeds are needed to better understandthe overall potential cardiovascular health and menopausal symptombenefits.

Funding SourcesThis study was funded internally by the Department of Nutrition

and Food Sciences, Texas Woman’s University.



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FIGURE P08-074-1 Systolic and diastolic blood pressure frombaseline to 12 wk in the pumpkin seed oil and pumpkin seedgroups.

FIGURE P08-074-2 Total symptom score from baseline to 12 wkof the pumpkin seed oil and pumpkin seed groups.

FIGURE P08-074-3 Severity of hot flushes from baseline to 12 wkin the pumpkin seed oil and pumpkin seed groups.

Particle Size andProcessingAffect Almond Lipid Bioaccessibility(P08-075)

Giuseppina Mandalari,1 Mary Parker,2 Myriam M Grundy,2Terri Grassby,3 Antonella Smeriglio,1 Carlo Bisignano,1 RobertoRaciti,1 Domenico Trombetta,1 David J Baer,4 and Peter JWilde2

1University of Messina, Italy; 2Quadram Institute Bioscience;3University of Surrey, UK; and 4USDA

Objectives: The aims of the present work were as follows: 1) toinvestigate the mechanisms responsible for the loss of observed in vivometabolizable energy from almonds compared with that calculatedfrom nutrient composition through the use of the Atwater generalfactors; 2) to carry out microstructural investigations on freshly,artificially masticated almond samples, to determine the particle sizedistribution of the masticated samples, and the extent of lipid releaseafter oral digestion; and 3) to further validate the mathematical modelpreviously developed to predict lipid release from masticated almonds.

Methods: The lipid released during simulated mastication from4 almond meals—natural raw almonds (NA), roasted almonds (RA),roasted diced almonds (DA), and almond butter from roasted almonds(AB)—was quantified by n-hexane extraction. Lipid release was pre-dicted based on a combination of themeasured particle size distributionby sieving, and the average cell diameter, 36 µm. Following simulatedoral digestion, the particle size of the samples before (AB) and after (NA,RA, DA, andAB) wasmeasured bymechanical sieving.Microstructuralanalysis of fecal samples from volunteers consuming NA, RA, DA, andAB was performed.

Results: Lipid release after mastication was 8.9% from NA, 11.8%from RA, 12.4% from DA, and 6.2% from AB. Close agreementwith the measured lipid release was obtained with NA, RA, and AB.The measured lipid release was higher than the predicted release(9.6%) in DA: this is probably the effect of roasting. The total lipidpotentially available for digestion in AB was 94.0%, which includedthe freely available lipid resulting from the initial sample processingand the further small amount of lipid released from the intact almondparticles during mastication. Particle size distributions measured after


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mastication in NA, RA, and DA showed most of the particles had asize of ≥1000 µm, whereas AB bolus mainly contained small particles(<850 µm). Microstructural analysis confirmed that some lipid in NA,RA, and DA remained encapsulated within the plant tissue throughoutdigestion, whereas almost complete digestion was observed in the ABsample.

Conclusions: The structure and particle size of the almond mealsare the main factors regulating lipid bioaccessibility in the gut.

Funding SourcesThis work has been funded by the United States Department of

Agriculture, Almond Board of California (USA) and the University ofMessina (Italy).

Exploring the Effects of Morinda citrifolia L. Fruit Extractin a Diabetes-Induced Retinopathy Animal Model Based on aMetabolomics Approach (P08-076)

Zulfitri Azuan Mat Daud, Aziatul Natasha Ahmad, Nazri Omar,Faridah Abas, and Amin Ismail

Universiti Putra, Malaysia

Objective: The aim of this study was to investigate the potentialmechanism of Morinda citrifolia L. (MC) fruit extract on glycemiccontrol and antidiabetic retinopathy through the use of a proton nuclearmagnetic resonance (1H-NMR)–based metabolomics approach.

Method: Fortymale Sprague-Dawley rats were divided into 5 groups[Control; Diabetic (DB); DB treated with 200 mg/kg ethanolic MC ex-tract (DB + MC200); DB treated with 400 mg/kg ethanolic MC extract(DB + MC400); and DB treated with 5 mg/kg gliclazide (DB + DG)]and were subjected to 8 wk of treatment. Glycemic responses weremonitored on a weekly basis, via blood sampled from the tail vein.Retinal examination was conducted through a dilated pupil with theuse of a direct ophthalmoscope and a portable slit lamp biomicroscope.Serum obtained when the rats were killed was analyzed for glucose andlipid profiles, and subjected to 1H-NMR–based metabolomics profilingbased on an untargeted approach. Unsupervised principal componentanalysis (PCA) was used to discriminate the serum metabolomicsprofile of different rat groups and to explore the change in associatedbiochemical pathways. The relative concentration of the metabolitesresponsible for the different profiles was quantified with the use of theChenomx NMR suite.

Results: The results obtained indicated that MC400 and MC200extracts were equally as effective as gliclazide in term of glycemiccontrol. At the end of 8 wk, 86% (n = 6) of animals in the DBgroup had moderate and severe diabetic cataract, as opposed to 50%(n = 4) in the DB + MC200; 25% (n = 2) in the DB + MC400group and 50% (n = 4) in the DB + DG group had only mild diabeticcataract. None of the animals in the control group developed cataract.PCA score plots showed clustering of the rat in the first and secondprincipal components, with separation between DB, DB + treatmentgroups, and control. DB + MC400 rats had metabolic perturbationclosest to control rats, suggesting the effectiveness of MC extract asan antidiabetic agent. Quantitative analysis showed that DB + MC200,DB + MC400, and DB + DG caused varying significant reductionsof glucose, 3-hydroxybutyrate, acetone, and acetoacetate, but elevated

levels of acetate, pyruvate, succinatem and alanine when comparedwith DB.

Conclusion:MC fruit extract administered at 400 mg/kg is effectiveas a hypoglycemic agent and delayed diabetic retinopathy in diabetic-induced rats, thus warranting further investigation in a clinical setting.

Funding SourcesThis research has been funded by NKEA Research Grant Scheme

(NRGS) under the Ministry of Agriculture Malaysia.

Freeze-Dried Grape Powder Improves Plasma Lipids and Mark-ers of High-Density Lipoprotein Function in Distinct Subsets ofMetabolic Syndrome Adults (P08-077)

Courtney L Millar, Quinn Duclos, Chelsea Garcia, Gregory HNorris, Bruno Lemos, Diana DiMarco, Maria-Luz Fernandez,and Christopher Blesso


Objective: High-density lipoprotein (HDL) particles protect fromatherosclerosis via reverse cholesterol transport, as well as throughantioxidant and anti-inflammatory properties. However, HDL functionis impaired under chronic inflammation, as seen inmetabolic syndrome(MetS). We evaluated the effects of grape ingestion on HDL functionalmeasures in adults with MetS.

Methods: Twenty adults with MetS were asked to consume either60 g/d of freeze-dried grape powder (GRAPE), equivalent to 2.5 cupsof fresh grapes daily, or a placebo for 4 wk in a randomized, double-blind, crossover design study, separated by a 3-wk washout period. Atthe end of each period, fasting bloodwas collected for analysis of plasmalipids, HDL size and subfractions, apolipoprotein A-I (apoA-I), serumamyloid A (SAA), metabolic markers, HDL cholesterol efflux capacity,and serum paraoxonase-1 (PON1) activities. We conducted analyseson the effects of GRAPE compared with placebo in all subjects, as wellas subgroup analyses in those below or above median values for HDLcholesterol (40 mg/dL, High HDL) and HDL cholesterol efflux capacity(Low Efflux or High Efflux).

Results: GRAPE consumption did not significantly alter metabolicmarkers, HDL particle concentrations, HDL size, apoA-I, or PON1activities compared with placebo in all subjects. However, in subjectswith Low HDL, GRAPE resulted in a lower plasma total choles-terol/HDL ratio (P = 0.04) and triglycerides (P = 0.04) comparedwith placebo. Compared with Low HDL, those with High HDLactually had increased markers of proinflammatory HDL across bothintervention arms. Subjects with High HDL had improved HDLcholesterol efflux capacity (P = 0.04) and PON1-lactonase/HDL-SAAratios (P = 0.06) with GRAPE. In those individuals with Low Efflux,GRAPE resulted in greater HDL cholesterol efflux capacity (P = 0.03)and PON1/HDL-SAA ratios (P = 0.03) compared with placebo.Overall, HDL functional measures were positively correlated with eachother, and inversely correlated with measures of insulin resistance andinflammation.

Conclusions:Grape consumption may improve plasma lipids, HDLcholesterol efflux capacity, and HDL anti-inflammatory potential indistinct subsets of MetS.

Funding SourcesThis work was supported by an award to CB from the California

Table Grape Commission.


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Blood Pressure–Lowering Effect of Doenjang Compared withHigh-Salt Intake in SD Rats (P08-078)

Eun-Gyung Mun,1 Vijaya Abinaya Ravichandran,2 Ha-YoungWoo,1 Suk-Heung Oh,2 and Youn-Soo Cha,1

1Chonbuk National University, South Korea; 2Department of FoodScience and Human Nutrition

Objectives: Korean fermented foods, including doenjang, contain alarge amount of salt. Salt is known to aggravate health problems, butfermented foods have a good effect. We hypothesized that doenjangcould lower blood pressure in SD rats fed a high-salt diet.

Methods: Eighteen male SD rats were divided into 3 groups and fedfor 8 wk as follows: normal diet group (ND), high-salt diet group (HS),and doenjang diet group (DJ). The salinity of doenjang and the saltwater fed by oral administration was adjusted to 8% byMohr’s method.The blood pressure of the rats was measured weekly through the useof the tail-cuff method. Serum rennin, angiotensin II, and aldosteroneconcentrations were analyzed with ELISA kits. The expression of genesinvolved in the renin-angiotensin system was measured by quantitativereal-time PCR.

Results: There was no significant difference in body, liver, andkidney weight among the groups. Blood pressure was decreasedsignificantly in DJ compared with HS and ND. Water intake andurinary excretion were significantly higher in HS than DJ. Excretionlevels of urine sodium, urine potassium, and fecal potassium weresignificantly increased in DJ compared with HS and ND. Serum reninlevels were significantly lower in DJ than in the other groups. Serumglutamic oxlaoacetic transaminase (GOT) and glutamate pyruvatetransaminase (GPT) levels were not significantly different amonggroups. Mineralocorticoid receptor (MR) expression in kidney cortexwas significantly higher in HS than in DJ. Na+/K+ ATPase α1 (NKA1)expression level of kidney cortex was significantly higher in DJ thanin HS.

Conclusions: Doenjang did not increase the blood pressures in SDrats despite its high salt content. These results suggest that soybean pastemay be effective in improving salt-related problems.

Funding SourcesThis research was supported by the Basic Science Research Program

through the National Research Foundation of Korea (NRF) funded bythe Ministry of Education (2015R1D1A3A01015869).

Vitamin D3 (calcitriol) Regulates Cancer and Macrophage CellFunction in The Breast Tumor Microenvironment In Vitro (P08-079)

Maliha Tabassum Munir,1 Julianna Santos,1 Zeina S Khan,1Kaiser Tarafdar,2 Fazle Hussain,1 and Shaikh Rahman1

1Texas Tech University; and 2Covenant Medical Center

Objectives: Breast cancer accounts for most cancer-related deathsin women in the United States. The breast tumor microenviron-ment (TME) consists of different cells including macrophages, whichcomprise ∼50% of the tumor mass. Accumulating evidence suggeststhat macrophages can be polarized into either M1 macrophages withantitumor properties or M2 macrophages with protumor activities in

response to TME signals. TAMs, usually of M2 phenotype, facilitatebreast tumor progression, angiogenesis, and metastasis by promotingimmunosuppression and chemoresistance. VitaminD (VD3) is essentialfor bone health and also has antitumor properties. We recently foundthat VD3 reduces glycolysis and cell invasiveness in breast cancer.The present study evaluated the effects of VD3 alone or togetherwith BEZ235 (PI3K/mTOR inhibitor) on macrophage and cancer cellfunctions in the TME.

Methods: MCF7- and THP1-derived macrophages were cul-tured individually and treated without and with VD3, BEZ235, andVD3 + BEZ235. The corresponding conditioned medium collectedfrom the macrophages were MCM, MCM + VD3, MCM + BEZ235,MCM+VD3 + BEZ235, and the cancer cells were CCM, CCM+VD3,CCM + BEZ235, CCM + VD3 + BEZ235. Cancer cells andmacrophages were induced with these conditioned media or theregular cell culture medium. Cells were harvested and processed forquantitative PCR, immunoblots, cell viability, and lactate assay. Datawere analyzed by Student’s t test or ANOVA.

Result: Macrophages treated with CCM demonstrated a decreaseof M1 (TNFα) and an increase of M2 (CD206) gene expressions,whereas CCM+VD3 and CCM+ BEZ235 showed the opposite effects.Interestingly, CCM + VD3 + BEZ235 treatment showed a more potenteffect than the individual treatments. MCF7 cells treated with MCMinduced the expression of proteins and genes implicated in glycolysis(HKII and LDHA) and in angiogenesis (matrix metalloproteinase9), increased cancer cell viability and lactate secretion, but theseeffects were suppressed by both MCM + VD3 and MCM + BEZ235treatments. In addition, MCM + VD3 + BEZ235 showed a synergisticeffect on MCF7 cells.

Conclusion: Our results indicate that the combined effects of VD3

and BEZ235 on macrophage and cancer cell functions in the TME aremore potent than the individual treatments and thus represent a newtherapeutic option.

Funding SourcesFunded by Start Up fund (TexasTech University) to SMR.

γ-Conglutin Hydrolysates from Andean Lupinus mutabilis SweetEnhance Glucose Uptake and Reduce Gluconeogenesis In Vitro(P08-080)

Erika Muñoz,1 Diego Luna-Vital,2 Marco Fornasini,2 ManuelBaldeón,2 Elvira Gonzalez de Mejia,1

1Universidad Tecnológica Equinoccial, Ecuador; and 2University ofIllinois

Background: Animal and human studies indicate that lupin seedconsumption has positive effects on the hyperglycemia and insulinresistance present in type 2 diabetes. There is limited informationregarding the molecular mechanism by which lupin proteins decreaseserum glucose levels.

Objective: This study aimed to assess the mechanism of actionof protein hydrolysates from Andean Lupinus mutabilis in glucosemetabolism in vitro.

Methods: Protein hydrolysates from defatted lupin flour wereobtained by pancreatin-pepsin digestion (semipurified γ -conglutin,


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SCgh) or pepsin-pancreatin digestion (simulated gastrointestinal di-gestion, SGDh); or from isolated γ -conglutin digestion (purified γ -conglutin, Cgh). The effects on glucose metabolism were evaluatedin a dual cell culture system of intestinal epithelial cells/adipocytesor intestinal epithelial cells/hepatocytes. In addition, confocal laserscanning microscopy depicting 2-dimensional fluorescence detectionand quantification determined by the intensity (AU) over area (μm2) ofglucose transporter type 4 (GLUT-4) in adipocytes after SCgh, SGDh,and Cgh treatment was performed.

Results: SCgh, SGDh, and Cgh at 5 mg/mL inhibited dipeptidylpeptidase IV activity (100%, 61.6%, 53.6%, respectively), and increasedglucose uptake (9.7 ± 0.3, 10.9 ± 2.9, 13.2 ± 0.8 mM, respectively)in comparison with untreated adipocytes (P < 0.01). Augmentedmembrane translocation ofGLUT-4 could explain the increased glucoseuptake upon lupin protein hydrolysate treatment. Cgh stimulationelicited greater translocation than SCgh or SGDh. In this model,insulin-induced glucose uptake was potentiated by addition of L.mutabilis hydrolysates. In addition, L. mutabilis hydrolysates decreasedgluconeogenesis by hepatocytes (54.4%, 59.6%, and 57.8%, respectively)in comparison with the untreated cells (P < 0.01), and reducedphosphoenolpyruvate carboxykinase expression.

Conclusion: L. mutabilis protein hydrolysates could affect glucosemetabolism by inhibitingDPP-IV enzymatic activity, improving insulinreceptor sensitivity and inhibiting hepatic gluconeogenesis. These re-sults strengthen the recommendation for the consumption of legumes,as part of the traditional diet, for the general population.

Funding SourcesUniversity of Illinois International Programs and Universidad

Tecnológica Equinoccial.

Polyphenol-Rich Extracts from Different Plums Decreasethe Inflammation in Lipopolysaccharide-Stimulated RAW264.7Macrophages (P08-081)

Angela D Myracle and Xue Du

University of Maine

Objective: Plums are polyphenol-rich fruits, which exhibit anti-inflammatory activity and thus could be beneficial in lowering the riskof chronic diseases when incorporated into the diet. The objective ofthis study was to quantify the phenolic compounds in 6 varieties ofplums, evaluate their antioxidant capacity, and investigate their anti-inflammatory potential through the use of RAW264.7 macrophages.

Methods: Freeze-dried plums (Oblinya, Vanier, Methley, RosyGage, Superior, and Caselton) were extracted with acidified 80%methanol. Folin-Ciocalteu assay was used to quantify the total phenoliccompounds. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay was usedto measure the antioxidant capacity. The anthocyanin content ofthe plums was measured with the pH differential method. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay wasconducted to test the cytotoxicity of plum extracts on RAW264.7macrophages at different levels. The anti-inflammatory effects of plumextracts were measured through the use of lipopolysaccharide (LPS)-inducedmacrophages, and the production of nitric oxide wasmeasuredvia Griess assay.

Results: Oblinya and Superior contained the highest amount oftotal phenolic compounds (13.4 and 13.5 mg gallic acid/g plum,respectively). Caselton had the lowest total phenolic content (6.3 mggallic acid/g plum), but it had the highest anthocyanin content (1.75mg cyanidin-3-glucoside/g plum), followed by Oblinya and Methley(1.69 and 1.57 mg cyanidin-3-glucoside/g plum). Oblinya and Superiorexhibited the highest antioxidant capacity and Caselton the lowest.Cytotoxic effects were not observed at 4 mg/mL. Superior (4 mg/mL)caused the highest inhibition (51.2%) of nitric oxide production inLPS-stimulated RAW264.7 cells, followed by Oblinya (27.7%), Methley(26.0%), and Vanier (25.6%) at the same concentration.

Conclusions: Polyphenol-rich plum extracts demonstrated anti-inflammatory activity, and the strongest activity was observed for theplum variety Superior.

Funding SourcesMaine Department of Agriculture, Conservation and Forestry.

Milk Sphingomyelin and its Hydrolytic Products InhibitLipopolysaccharide Activity in RAW 264.7 Macrophages (P08-082)

Gregory H Norris, Caitlin Porter, Christina Jiang, and Christo-pher Blesso


Objective: Dietary sphingomyelin (SM) and other sphingolipidshave been studied for their effects on dyslipidemia due to theirinhibition of dietary fat and cholesterol absorption. In addition to effectson lipid absorption, dietary SM may have further bioactive effects byreducing inflammation. We aimed to determine whether milk SM andits hydrolytic products (ceramides, dihydroceramides, and sphingosine)directly affect the inflammatory response of macrophages stimulatedwith lipopolysaccharide (LPS).

Methods: Murine RAW 264.7 macrophages were incubated with1 ng/mL LPS with and without milk SM, ceramides, or sphingosine(>99% purity) for 4 h. Total RNA from cells was isolated, synthesizedinto cDNA, and then real-time quantitative reverse transcriptase-polymerase chain reaction analyses were conducted for expressionof proinflammatory genes (Tnf, Ccl2). To determine if effects weredependent on SM hydrolysis, some cells were also pretreated withimipramine for 2 h to inhibit sphingomyelinase before the addition ofLPS and SM.

Results: LPS-stimulated increases in Tnf mRNA and Ccl2 mRNAwere significantly attenuated with the addition of milk SM (0.8and 8 μg/mL). In the absence of LPS, milk SM did not influenceproinflammatory gene expression. Cell viability was unaffected by milkSM at the concentrations tested. With the addition of imipramine,LPS-stimulated inflammatory gene expression was unaltered by milkSM. Both C16-ceramide and C24-ceramide (10 μM) reduced TnfmRNA and Ccl2 mRNA in LPS-stimulated macrophages, but theircorresponding dihydroceramides did not. None of the ceramides testedaltered inflammatory gene expression in the absence of LPS. Theobserved effects of ceramides (sphingosine base), but not dihydroce-ramides (sphinganine base), suggested that sphingosine (d18:1) wasimportant for bioactivity. In support of this, sphingosine (1–10 µM)significantly reduced LPS stimulation of Tnf mRNA and Ccl2 mRNA in


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macrophages. Neither ceramides nor sphingosine significantly affectedcell viability or caspase 3 activity at the concentrations tested, indicatingthat cell death was not the cause of anti-inflammatory effects.

Conclusion: Our data suggest that milk SM may be effective inreducing inflammation through inhibition of LPS activity and that thehydrolytic products of SM are important for these effects.

Funding SourcesThis work was supported by an award to CB from the University of

Connecticut’s Research Excellence Program.

Amylopectin/Chromium Complex (ACr; Velositol): Liver andKidney Function Safety Data (P08-083)

Sara Perez Ojalvo,1 Kazim Sahin,2 Sarah Sylla,1 and JamesKomorowski1

1Nutrition 21, LLC; and 2Firat University, Turkey

Objectives: Prior research has shown that when added to wheyprotein (WP), an amylopectin/chromium complex (ACr; Velositol)significantly enhances muscle protein synthesis (MPS) in humanskeletalmuscle. This preclinical studywas conducted to evaluateMPS inrats administered different forms of protein, with and without Velositol.Because high-protein diets have been associated with the potentialrisk for impairing liver and kidney function, blood samples from thispreclinical studywere analyzed to evaluateVelositol’s effects on liver andkidney function.

Methods: Male Wistar rats (8 wk old) were assigned to a controlgroup, or one of several protein groups (8 rats/group). Rats received:WP (0.465, 1.55, 2.33, or 3.1 g/kg body wt; a 6, 20, 30, or 40 g

FIGURE P08-083-1

human dose equivalent (HED)), branched-chain amino acids (0.465g/kg body wtW; a 6 g HED), or pea protein (0.465 g/kg body wt; a6 g HED) with or without Velositol (1.55 g/kg body wt; a 2 g HED).As part of the MPS study, all rats were exercised with a treadmillfor 10 d pretreatment. On the day of the single-dose experiment, ratswere exercised at 26 m/min for 2 h and then fed their assigned studyproduct via oral gavage immediately after exercise. One hour later, theanimals were killed, and blood samples were taken to analyze markersof liver and kidney function [aspartate transaminase (AST), alanineaminotransferase (ALT), and creatinine (CREA)].

Results: Compared with the protein-alone groups, there were nosubstantial changes in AST, ALT, or CREA levels in the Velositolplus protein groups. However, there was a slightly larger decreasein AST levels, indicating improved liver function, in the Velosi-tol plus protein groups compared with the protein-alone groups(Figure 1).

Conclusions: In this preclinical study, Velositol did not showany detrimental effects on liver and kidney function when addedto various sources and doses of protein. Rather, a greater decreasewas seen in AST levels in Velositol plus protein groups, suggestingthat Velositol intake with protein after exercise promotes healthy liverfunction. This study provides additional data supporting the safeuse of Velositol in individuals consuming higher levels of protein,enabling them to gain strength while also maintaining liver and kidneyhealth.

Funding SourcesThis study was funded by Nutrition 21, LLC.



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The Effect of a High-Fat Meal Containing Culinary Doses ofSpices on Postprandial Endothelial Function, Lipids and Lipopro-teins, and Glucose (P08-084)

Kristina S Petersen, Sheila West, Ester Oh, Connie J Rogers,Penny M Kris-Etherton

Pennsylvania State University

Objective: Including large doses of spices (>11 g/meal) in a high-fat meal blunts postprandial triglyceride levels, which may reducevascular impairment. The effect of lower quantities of spices, found inmany meals, has not been investigated. Our aim was to examine theeffect of a high-fat meal containing culinary doses of herbs and spiceson postprandial endothelial function, lipids/lipoproteins, and glucoselevels.

Methods: A 3-period randomized controlled crossover study wasconducted. In random order, subjects consumed either a high-fat meal(1000 kcal, 45 g fat) or a high-fat meal containing 2 g of spices or a high-fat meal containing 6 g of spices. After meal consumption, blood wasdrawn hourly for 4 h and flow mediated dilation (FMD) was measuredat 2 and 4 h. Participants (n = 10) were overweight/obese, nonsmokingmales with increased waist circumference (≥94 cm) and had at least oneother cardiovascular risk factor.

Results: A borderline treatment (P = 0.057) and time (P = 0.055)effect existed for FMD, the primary outcome. Exploratory analysisshowed this treatment effect was due to higher FMD values after themeal with 6 g of spices compared with the control meal with no spices(mean difference 0.94 ± 0.39%; P = 0.049); no significant time bytreatment effect existed (P = 0.85). In all three treatment groups, thehigh-fat meal markedly increased serum triglycerides, compared withfasting values (time effect P < 0.001); triglyceride response was notmodified by treatment (P = 0.167). Similarly, plasma glucose levelsincreased from fasting in all treatments (time effectP< 0.001); however,a treatment effect (P = 0.031) existed such that average glucose levelsfollowing the meal with 2 g of spices were lower than the control meal(107 ± 3.1 compared with 114 ± 3.74 mg/dL; P = 0.029). A treatmenteffect was also observed for total cholesterol (P = 0.044), low-densitylipoprotein cholesterol (P= 0.011), high-density lipoprotein cholesterol(P = 0.016), and the cholesterol to HDL ratio (P < 0.001). The totalcholesterol to high-density lipoprotein cholesterol ratio was lower afterboth the 2- and 6-g meals compared with the control meal (P < 0.01).

Conclusion: These preliminary analyses suggest that inclusion ofherbs and spices in a high-fat meal attenuates postprandial FMDimpairment, but improves lipids and lipoproteins and plasma glucoseprofile.

Funding SourcesMcCormick Science Institute.

Hepatoprotective Effects of a Standardized Pomegranate Ex-tract on High-Fat-Diet–Induced Nonalcoholic Fatty Liver Disease(NAFLD) in Mice (P08–085)

Marisa Pfohl, Nicholas DaSilva, EmliyMartell, HangMa, AngelaL. Slitt, Navindra Seeram

University of Rhode IslandObjective: Nonalcoholic fatty liver disease (NAFLD) is a common

chronic liver disorder that is characterized by abnormal accumulation of

triglycerides (TG) in liver cells. Despite the high prevalence of NAFLDin the general population of Western countries (20–30%), limitedpharmacologic agents have been approved for its treatment. Severalstudies suggest that natural products including dietary polyphenolsshow beneficial effects against NAFLD. Herein, we evaluated thehepatoprotective effects of a standardized pomegranate extract (PE) inmice with high-fat-diet–induced NAFLD.

Methods: The 6-wk-old C57BL/6 male mice were divided into 4groups (n= 4) and fed with low-fat diet (LFD; 10% kcal as fat), high-fatdiet (HFD; 45% kcal as fat), LFD with PE (1% wt/wt in diet), or HFDwith PE (1% wt/wt in diet) for 12 wk.

Results:Mice in both the LFD and HFD groups with PE had lowerliver TG levels by 30.0% and 60.2%, as comparedwith the LFD andHFDgroups, respectively. In addition, the HFD + PE group significantlyreduced the mice body weight by 23.3% as compared with the HFDgroup. The HFD + PE group also had reduced liver lipids and livernonesterified fatty acids levels by 41.8% and 70.8%, respectively, ascompared with the HFD group.

Conclusions: These results suggest that PE may be beneficialfor the amelioration of NAFLD, warranting further evaluation of itshepaprotective effects.

Evaluationof Spirulina platensis Supplementation for thePreven-tion of Liver Inflammation and Fibrosis (P08-086)

Tho X Pham, Minkyung Bae, Yoojin Lee, Siqi Hu, NicholasSantangelo, Mi-Bo Kim, Hyunju Kang, Young-Ki Park,and Ji-Young Lee


Objective: The incidence of nonalcoholic fatty liver disease(NAFLD) has increased with the rise of the obesity epidemic. Liverfibrosis presents a particular challenge as there is currently no effectiveanti-fibrotic treatment. Spirulina platensis (SP) is an edible blue-greenalga that is widely added as a supplement in health foods. SP hasbeen demonstrated to possess anti-inflammatory and hepatoprotectiveeffects. The objective of this study was to determine whether supple-mentation with SP can prevent obesity-induced hepatic inflammationand fibrosis in vivo.

Methods: Forty-five male C57BL/6J mice were randomly assignedto a low-fat diet (LFD) or a high-fat/high-sucrose/high-cholesterol diet(HFD), or HFD supplemented with 2.5% wt/wt SP (SPD) for 20 wk.Plasma was collected every 4 wk and mice were subjected to oralglucose tolerance test (OGTT) at week 18 and indirect calorimetryat week 19. Body weight and tissue weights were measured. TissuemRNA expression for markers of inflammation and fibrosis wasanalyzed.

Results: There were no differences in the body weight of miceon HFD or SPD throughout the study. Indirect calorimetry analysisshowed that there were no differences in the activity or energyexpenditure between mice on HFD and SPD. As measured by OGTT,blood glucose levels were significantly lower in mice on the SPD at60 min compared to the HFD, but not at 120 and 180 min. Plasmaconcentrations of alanine aminotransferase (ALT) and triglyceride,but not total cholesterol, were significantly reduced after 16 week inSPD mice compared with HFD mice. At week 20, however, serum


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triglyceride, cholesterol, and ALT levels were not different betweenmice on HFD or SPD. Liver gene analysis revealed that there wereno differences in the expression of markers of liver fibrosis andinflammation between HFD and SPD. Basal expression of interleukin(IL)-1b, but not tumor necrosis factor α and IL-6, was significantlyreduced in the splenocytes isolated from mice on SPD comparedwith HFD.

Conclusion: SP supplementation appears to attenuateHFD-induceddyslipidemia and liver damage early on in C57BL/6 mice, but did notattenuate HFD-induced hepatic inflammation and fibrosis by 20 wk.Further studies with different feeding periods are required to fullyevaluate whether SP may be effective in preventing obesity-inducedhepatic inflammation and fibrosis.

Funding SourcesUSDA NIFA (2016-04624).

Sucrose- and Doxorubicin-based Chemotherapy Affect BrainFatty Acids in Mice Fed an ω-3 Fatty Acid–Enriched Diet (P08-087)

Panchita Phuwamongkolwiwat-Chu, Monica Gaudier-Diaz, Re-becca Andridge, Maryam B Lustberg, Courtney DeVries, andTonya Orchard


Objective: Doxorubicin-based chemotherapy for breast cancer canincrease neuroinflammation and may lead to long-term cognitivedeficits. Although increasing ω-3 fatty acid (n–3 FA) intake isa promising strategy to reduce neuroinflammation and minimizenegative cognitive outcomes, limited data are available on the effects ofthe background diet on the incorporation of n–3 FAs in the brain duringchemotherapy. We hypothesized that a low sucrose diet enriched withn–3 FAs [e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA)]would significantly increase brain n–3 FAs in a postmenopausalmouse model of chemotherapy.

Methods: Female C57BL/6 mice (8–9 wk old) were ovariectomizedand then randomly assigned to 1 of 4 diets: low sucrose or high sucrosewith 2% kcal EPA + DHA or no EPA + DHA. After 2 and 4 wkof diets, mice received a saline or chemotherapy injection (9 mg/kgdoxorubicin + 90 mg/kg cyclophosphamide). Mice were killed 2 wkafter the second injection; their brains were collected and snap-frozenin liquid nitrogen. Fatty acids in brain were extracted, methylated, andanalyzed by gas chromatography. Significant differences (P< 0.05) weredetermined by 3-way ANOVA.

Results: Levels of n–3 and n–6 fatty acids in mouse brainshowed significant differences among diets. The n–3 fatty acids, EPA(20:5n–3), docosapentaenoic acid (22:5n–3), and DHA (22:6n–3) weresignificantly higher in 2% EPA + DHA diet groups (P < 0.001 forall). In contrast, n–6 fatty acids, including linoleic acid (LA; 18:2n–6),eicosadienoic acid (20:2n–6), and arachidonic acid (AA; 20:4n–6), weresignificantly higher in the no EPA + DHA diet groups (P < 0.0004 forall). Additionally, there was a significant main effect of sucrose on LA,such that LAwas significantly higher in the low -sucrose compared withthe high-sucrose groups (P = 0.01). There was a significant sucrose byinjection interaction on DHA such that, in chemotherapy groups, brainDHA was significantly higher in the low-sucrose groups than in thehigh-sucrose groups (P = 0.009).

Conclusion:Our findings suggest that dietary sucrose, doxorubicin-based chemotherapy, and EPA + DHA intake alter fatty acid profilesin brain. Future studies should investigate the combination effect ofhigh levels of EPA + DHA with low sucrose on chemotherapy-inducedcognitive deficits.

Funding SourcesNIH: National Cancer Institute.

An ω-3–Enriched Diet Decreases Chemotherapy-Induced In-flammation in a Mouse Model (P08-088)

Panchita Phuwamongkolwiwat-Chu, Monica Gaudier-Diaz, Re-becca Andridge, Maryam B Lustberg, Courtney DeVries, andTonya Orchard


Objective: Doxorubicin-based chemotherapy results in long-termcognitive deficits in ∼30% of cancer survivors who receive the treat-ment. Increased neuroinflammationmay be a contributingmechanism.Increasing ω-3 fatty acid (n–3 FA) intake is a promising strategyto reduce inflammation in the brain. Thus, we hypothesized thata diet mixture of n–3 FAs [e.g., eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA)] and low sucrose would significantlyincrease the concentration of n–3 FAs and decrease chemotherapy-induced inflammatory cytokines in the brain.

Methods:Adult, female, ovariectomized BalbCmice were randomlyassigned to 1 of 2 diets: either low sucrose diet with 2% kcalEPA + DHA (Low Suc, 2% EPA + DHA) or high sucrose withoutEPA + DHA (High Suc, no EPA + DHA). The mice received 2saline or chemotherapy injections (75% human equivalent dose ofdoxorubicin and cyclophosphamide), administered 1 wk and 3 wk afterstarting diets. Mice were killed 6 h after the second injection; theirbrains were collected and frozen at –80°C. One brain hemisphere wasanalyzed for gene expression of inflammatory cytokines [tumornecrosisfactor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6)] byquantitative PCR. The other hemisphere was analyzed for fatty acids bygas chromatography. Significant differences (P< 0.05) were determinedby 2-way ANOVA.

Results: Brain EPA and DHA levels were significantly greater(P< 0.05 for both) inmice fed Low Suc, 2% EPA+DHAdiet comparedwith High Suc, no EPA + DHA. There was a significant diet effecton IL-1β in cortex such that IL-1 β was lower in the Low Suc, 2%EPA+DHAgroup (P= 0.0013). There was a significant injection effecton hippocampal cytokines: in the chemotherapy group, IL-1β was lower(P= 0.0057) and IL-6 was higher (P= 0.252). No significant differenceswere seen in expression of TNF-α.

Conclusion: Our findings suggest that when brain EPA and DHAare increased, IL-1β , a marker of inflammation, can be decreased afteronly 3 wk of feeding a diet high in n–3 FAs and low in sucroseduring doxorubicin-based chemotherapy treatment. Further studies ofthe anti-inflammatory and neuroprotective potential of administeringn–3 FAs during chemotherapy is warranted.

Funding SourcesNIH: National Cancer Institute.


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Vitamin D3 (Calcitriol) Regulates Cancer Cell Proliferation, An-giogenesis and Cholesterol Metabolism via Epigenetic Modification(P08-089)

Christopher Ponce,MalihatabassumMunir, Hazera Binte Sufian,and Shaikh Mizanoor Rahman

Texas Tech University

Objectives: Breast cancer is the second most-common cancer-related death in the United States. Cancer cells undergo metabolicchanges during progression. Several studies have revealed cholesterolas an important regulator of breast cancer development, althoughthe molecular mechanisms remain unknown. Epigenetic changes canactivate or silence gene expression and play critical roles in breasttumorigenesis Vitamin D is not only an essential component for boneand calcium metabolism but shows anticancer property. However,whether vitamin D regulates cancer cell proliferation and angiogenesisby regulating cholesterol metabolism and epigenetic modification isnot known. We hypothesize that vitamin D prevents cancer cellproliferation and angiogenesis by regulating cholesterol homeostasisand epigenetic changes.

Methods:MCF7 [estrogen receptor (ER)-positive] cells were grownin high-glucoseDMEMand treatedwith (0.5, 1, and 2mM) andwithout(control) vitamin D3 (VD3, calcitriol) for 24 h. Cells were harvestedand lysed with lysis buffer, and nuclear and cytosolic fractions wereprepared. Western blot and quantitative PCR were employed to analyzeprotein and gene expression. Student’s t test and ANOVA were used forstatistical analysis

Results: VD3 treatment significantly increased the expression ofLXRα (which regulates cholesterol metabolism), ATP-binding cassettetransporter A1 (ABCA1, a protein involved in cholesterol efflux)and 5′AMP-activated protein kinase (AMPK, a regulator of energymetabolism and an inhibitor of inflammation) activity as evaluatedby identifying the expression of phosphorylated AMPK (P-AMPK)and cleaved caspase 3 (which is involved in apoptosis) proteins.Interestingly, VD3 treatment also decreased cancer cell viability andthe expression of the gene (matrix metalloproteinase 9) implicatedin angiogenesis. Importantly, DNA methyl transferase 1 (DNMT1),which is responsible for DNA hypermethylation and breast cancercarcinogenesis, is significantly decreased by VD3 treatment in MCF-7cells.

Conclusion: The results indicate that VD3 regulates MCF-7 prolif-eration, angiogenesis, and cholesterol metabolism in part via epigeneticregulation, and may be an important therapeutic option to treat ER-positive breast cancer.

Funding SourcesTexas Tech University start-up fund.

California Date Varieties are Rich in Bioactive CompoundsFibers and Proanthocyanidins (P08-090)

Charlene J Rainey,1 Emilia Alfaro-Viquez,2 Christian GKrueger,2 and Jess D Reed2

1Date Research Institute; and 2University of Wisconsin, Madison

Deglet Noor and Medjool are the two main varieties of Californiadates. These differ in texture, taste, size, and composition. The objective

of this study was to analyze the composition of bioactive componentsof both varieties of fresh, ripe dates for phenolic compounds, fibers,and composition. AOACmethods of analysis forminerals and nutrientswere used; polyphenols were identified by positive reflectron modematrix assisted laser desorption ionization-time of flight massspectrometry and reversed phase HPLC with diode array detection.

The proanthocyanidins (PACs) found in dates can be partitionedinto two categories: soluble (extractable) and insoluble (nonextractable)PACs. Insoluble PACs are bound to plant cell wall components suchas fiber or protein. Differences in polyphenol and fiber content andcomposition may influence sensory attributes of dates, such as fruitsoftness and integrity. Both soluble and insoluble PACs may beconsidered bioavailable, as these components interact with the gut-associated lymphoid tissue (GALT), intestinal epithelial surfaces, andintestinal microflora during the digestive process. Our research showsthat the major polyphenol compounds in dates were hydroxycinnamicacids, specifically, caffeic, p-coumaric, and ferulic acids, as well ascinnamic acid derivatives. The soluble PACs found in dates are mostlythose with B-type interflavan bonds. Recent research has demonstratedthat date polyphenols (inclusive of PACs) show bioactivity throughmodulation of the nuclear receptor (famesoid x receptor) which isresponsible for maintaining triglyceride and cholesterol homeostasis.Suberin compounds, specifically an ester of ferulic acid with long-chain hydroxyfatty acids (C16, C18), are another class of polyphenolderivatives which have recently been characterized in dates. Suberinsare waxy substances present in cell walls which also may influence fruitstructure and impart bioactivity. Deglet Noor and Medjool varietiesof dates were analyzed for nutrients and found to be similar inwater content (20.2% and 20.3%, respectively). Some differences inminerals may indicate types of fiber structures. Calcium in Medjoolswas slightly higher than in the Deglet Noor (52.8 compared with 39.6mg/100 g, P < 0.0005). Detailed analyses are needed to determineif calcium is an indicator of pectin structures. Potassium values forMedjools were higher than for Deglet Noor (675 compared with554 mg/100, P

Funding SourcesTheCaliforniaDateCommission provided funding for this research.

δ-Tocotrienol and Tart Cherry Anthocyanins Reduce Inflamma-tion in 3T3-L1 Adipocytes (P08-091)

Latha Ramalingam, London Allen, Shane Scoggin, Shasika Uda-hawatte, Iurii Kobozeiv, Chwan-Li Shen, and Naima Moustaid-Moussa

Texas Tech University

Objectives:Obesity is a disease that affects approximately one-thirdof American adults, and one of its major underlying causes is inflam-mation. Various food bioactives have anti-inflammatory properties. Weare specifically interested in δ-tocotrienol (dT3), an isoform of vitaminE, and tart cherries, which are rich in anthocyanins (TCAs). We havepreviously reported high-fat-diet–induced obese mice consuming dT3have reduced fat cell size and adipose tissue inflammation. However,themechanisms behind these findingswere not studied.Moreover, TCAalso exerts anti-inflammatory effects in vitro and in vivo. Although bothdT3 and TCA possess anti-inflammatory properties, their combined


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effects have not yet been determined. Hence, we are investigatingwhether dT3 and TCA reduce inflammation in adipocytes, and if thenuclear factor κ light-chain enhancer of activated B cells (NF-κB)pathway mediates these effects.

Methods: Clonal 3T3L1 preadipocytes and adipocytes were pre-treated for 4 h with dT3 with or without TCA, then stimulated with LPSplus dT3 with or without TCA for another 18 h. Cells were treated withup to 5 µMof dT3 and up to 16 µg/mL of TCA from TC extracts. Thesedoses were chosen based on previously established dose-response andcell-viability assays that use MTT assay. Total RNA and proteins wereisolated for gene and protein analyses. Western blotting was conductedto assess NF-κB activation via P65 phosphorylation.

Results: Doses of dT3 up to 25 µM were not toxic to preadipocytesor adipocytes, and none of the tested TCA doses were toxic. Inadipocytes, combinations of dT3 and TCA significantly reducedsecreted interleukin-6, more than individual treatments. Lower dosesof dT3 and TCA (1 µM of dT3 and 18 µg/mL of TCAs) resultedin the most significant reduction in inflammation among all dosecombinations tested, when compared with control. Similar resultswere obtained in preadipocytes. Moreover, with the above doses, TCAbut not dT3 significantly reduced lipopolysaccharide-induced P65phosphorylation.

Conclusions: dT3 and TCA exerts individual and combinatorialanti-inflammatory effects, which may be beneficial in treating orpreventing obesity. Additional experiments are ongoing to determinespecific pathways mediating these effects of dT3 and TCA, and themechanistic basis for their interactions.

Funding SourcesTexas Tech Universit startup funds.

25-Hydroxyvitamin D and the Intestinal Vitamin D Response(P08-092)

Carmen J Reynolds,1 Nicholas Koszewski,1 Ronald Horst,2Donald Beitz,1 and Jesse Goff1

1Iowa State University; and 2Heartland Assays

Objective: Hydroxylation of 25-hydroxyvitamin D3 (25-D) yieldsthe vitamin D hormone 1α,25-dihydroxyvitamin D3 (1,25-D). Thisactivation event occurs in the kidney; however, extra-renal expressionof 1α-hydroxylase (1α-OHase) in the intestines has been observed.This observation suggests that 25-D derived from the diet can elicithormonal effects within the intestines. We hypothesized that orallyadministrated 25-D will result in a local 1,25-D-response followingactivation by intestinal 1α-OHase.

Methods: We investigated the activity of 25-D in the intestinesby assessing the response to a single oral dose of 25-D or 1,25-D in mice. Responses were evaluated by the increased expressionof Cyp24 mRNA, a reporter gene for vitamin D receptor (VDR)stimulation. In situ hybridization (ISH) with RNAscope was performedto identify responsive cells in the intestine. An in vitro model withthe HT29 human colon cancer cell line was developed to mimic theapical enterocyte environment. Through the use of this model, wemanipulated the activity of 1α-OHase through ketoconazole inhibitionand CRISPR gene editing, then measured the responses to 25-D and1,25-D treatments.

Results: Mice that were orally administered with 25-D demon-strated increased expression of Cyp24 mRNA in the duodenum similarto, but less than, 1,25-D treatment. RNAISH revealed that the Cyp24mRNA responses from both treatments were localized to the epithelialcells of the intestines. In vitro, the enterocyte model enabled a responseto 100 nM 25-D treatment that was not observed under standard cellculture conditions. Concentration curve analysis comparing 1,25-Dwith 25-D revealed identical Vmax properties, but a difference in EC50

values of∼160-fold. Inhibition of 1α-OHase with the use of media with10 μM ketoconazole did not diminish the response to 1,25-D or 25-Dtreatments. Preliminary results with CRISPR knock-down of 1α-OHasesuggest no differences for Vmax and EC50 values with 1,25-D or 25-Dtreatments.

Conclusion: Although it is plausible that 25-D can be hydroxylatedto 1,25-D in the intestines, it is evident that 25-D elicits a responsewithout the need for 1α-OHase or 1,25-D synthesis. Thus, 25-D actsas a direct ligand agonist for VDR in the intestinal epithelium.

Funding SourcesNIH grant 2R15CA173628-02.

Do Antioxidant Treatments Affect DNA Methyltransferase Ex-pression in Murine Adipocytes? (P08-093)

Yeong Rhee

North Dakota State University

Background:DNAmethylation affects gene expression by changingthe level of transcription and mRNA synthesis. DNA methyltrans-ferases (DNMTs) are responsible for the methylation of DNAs,and hypermethylation of DNAs by DNMTs may affect physiologicfunctions, thus leading to chronic diseases. Oxidative damages toDNAs or cells caused by reactive oxygen species (ROS) also affectphysiologic functions. Additionally, ROS increases DNMT activities,thus leading to hypermethylation of DNAs. Therefore, neutralizationof ROS by antioxidants is important to prevent oxidative damages andhypermethylation of DNAs.

Objective: This study was conducted to measure the effects ofantioxidant treatments on DNMT gene expression.

Methods:Murine adipocytes (1× 107) were treated with myricetin,resveratrol, lipoic acid, α-tocopherol (0, 10, 50, 100 µM), or superoxidedismutase (100 U/mL, control antioxidant), and then treated withsuperoxide anion. After superoxide anion treatment, total RNA wasextracted and purified with an RNeasy Mini kit. DNMT1, DNMT3a,and DNMT3b gene expression by was measured real-time PCR. Thefold change in expression of the DNMTs was calculated through the useof the 2-��CT method.

Results: Resveratrol or myricetin (100 µM) treatment significantlydownregulated DNMT1 and DNMT3a expression, but did not affectDNMT3b expression. Lipoic acid or α-tocopherol treatment did notaffect DNMT1, DNMT3a, or DNMT3b expression.

Conclusions: Further study is needed to determine if resveratrolor myricetin also affects DNMT expression in adipocytes treatedwith different ROS, and to determine how resveratrol or myricetindownregulated DNMT expression in superoxide anion–treated murineadipocytes.


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Combinatorial Effect of Tocotrienols and Topoisomerase IIInhibitors on Breast Cancer Cells (P08–094)

Sandra Rizk,1 Maya Idriss,2 Mohamed Moustafa,2 and RajaaFakhoury2

1Lebanese American University, Lebanon; and 2Beirut Arab Univer-sity, Lebanon

Background: Etoposide, a Mayapple podophyllotoxin derivative,is a semisynthetic compound found to be effective against a broadspectrum of cancer types such as carcinoma of lungs, bladder, breast,gastric, testicular, acute myelocytic leukemia, and lymphoma. Despiteits potent antitumor activity, as a topoisomerase II poison, Etoposideis constrained by its low aqueous solubility and its clinical use islimited because of its adverse effects, mainly myelosuppression and thedevelopment of secondary leukemia. Trying to combine health and diet,and reducing etoposide toxicity, we aimed in this project to study themode of action of etoposide alongwith the potent antioxidant vitamin Emember, γ - tocotrienol (γ -TCT), reported to be a promising anticanceragent, on survival and apoptosis pathways in the breast cancer cell lineMDA-MB-231, and to investigate the potential synergistic effect of γ -TCT with etoposide.

Materials and Methods: MDA-MB-231 cells were incubated withdifferent etoposide and γ - TCT concentrations for 48 h, separately andin combination. Cell proliferation was determined with the use ofWST-1 (Roche) reagent. Apoptosis was assessed by cell death ELISA andannexinV/propidium iodide staining, and the cell cycle analysis wasperformed in addition to propidium iodide staining.

Results: Etoposide and γ -TCT exhibited antiproliferative effectson MDA-MB-231 cells when applied separately. In combination, thereduction in proliferation was extremely significant with a noticeableincrease in DNA fragmentation leading apoptosis. Moreover, thecombination induced a major S-phase arrest.

Conclusion: These fndings suggest that γ -TCT may be a usefulchemotherapeutic adjuvant to potentiate the pharmacologic action ofetoposide and ameliorate its adverse effects.

Funding SourcesSRDC, Lebanese American University, Beirut, Lebanon.

Effects of the Low- and High-Daidzein Diet on Bone Density andOsteogenic Gene Expression in FemaleObese Zucker Rats (P08-095)

Eric Rochester,1 Brooke Wickman,2 Changqi Liu,2 Andrea Bell,3Christy Bekkevold,3 Shirin Hooshmand,2 and Reza Hakkak,3

1San Diego State University School of Exercise and NutritionalSciences; 2San Diego State University; and 3University of Arkansas forMedical Sciences

Objective: Phytoestrogens, including isoflavones found in soybeansand other legumes, are nonsteroidal plant compounds with conspic-uously similar chemical structures to mammalian estrogen, and arethus capable of mimicking estrogen’s effects in selective tissues. A dietrich in phytoestrogens is associated with a variety of health benefitsincluding reduced symptoms of menopause, and decreased risks forheart disease, breast cancer, and osteoporosis in lean populations.Obesity and osteoporosis are two chronic conditions that have beenincreasing in prevalence in the United States and worldwide. Daidzein,

an isoflavone from soy, has been shown to improve bone health in leananimal models of osteoporosis but not in obese animals.

Methods: To investigate the effect of daidzein, a soy isoflavone,on bone health in an obese population, nineteen 5-wk-old femaleobese Zucker rats (OZR) were acclimatized for 1 wk on an AIN-93Gdiet, and were then randomly assigned to a modified AIN-93G dietcontaining either high daidzein (HD, 0.12 g/kg feed) or low daidzein(LD, 0.01 g/kg feed). After 8 wk, tibias and femurs were removedand true density measured with the use of Archimedes’ principle.Additionally, femoral mRNA expression of various genes related tobone health were measured by reverse transcription polymerase chainreaction.

Results: Our results indicated that there were no significant differ-ences between the tibial or femoral true density measures (P = 0.834and P = 0.299, respectively) of the rats in the HD and LD dietgroups. Similarly, there were no significant differences found in geneexpressions related to bone health, including RANKL (P= 0.439), OPG(P= 0.363), NOX4 (P= 0.429), BGLAP (P= 0.173), SOST (P= 0.909),DKK1 (P = 0.502), CTNNB1 (P = 0.931), WNT3A (P = .097), AXIN1(P = 0.485), RUNX2 (P = 0.761), and LRP5 (P = 0.990).

Conclusion: The results indicate daidzein in soy does not enhancebone health in an obese population.

Funding SourcesThe project was funded by the Arkansas Childrens Research

Institute’s University Medical Group Fund grant program.

Oral Glutathione Supplementation in Older Healthy Adults andits Effect on Immunity and Illness Symptoms During the Cold/FluSeason (P08-096)

Camila Rodrigues, Changjie Xu, Brandon J Eudy, Sawsan Abed,Alison H O’Donoughue, Carmelo Nieves, Cheryl Rowe, AnneMathews, Bobbi Langkamp-Henken, and Susan Percival

University of Florida

Objective: Glutathione (GSH) is a tripeptide that acts as the mainintracellular antioxidant, protecting cells against toxins, including freeradicals. GSH can protect host immune cells through its antioxidantmechanism.However, there are circumstances inwhichGSH is requiredin greater amounts, such as for elderly individuals or during times ofillness. The objective of this study was to evaluate the effect of an oralGSH supplement on the immune response and illness symptoms duringthe cold/flu season in older healthy adults.

Methods: Our sample comprised 102 generally healthy adults,aged 50–75 y, who completed all of the assessments and intervention.Participants must have had an episode of cold/flu during the past yearand agreed to discontinue the consumption of probiotics and dietarysupplements during the study. Immunocompromised individuals orindividuals taking immunosuppressive drugs were excluded from thestudy. Participants were instructed to consume 2 capsules daily of aproprietary formulation of GSH (500 mg/d) or placebo for a periodof 120 d. Subjects were asked to keep a daily diary of health andwell-being. Blood lipid profiles, lymphocyte populations, neutrophilrespiratory burst, cytokines, and cellular GSH levels were measuredfrom blood samples collected from fasting subjects at baseline and afterintervention.


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Results: After 120 d, male subjects receiving the GSH supplemen-tation showed a significant reduction in the systolic and diastolic bloodpressure. Furthermore, there was a significant increase in interleukin(IL)-6 and tumor necrosis factor α (TNFα) levels, by 22% and 17%,respectively, with GSH intake in males but not in females. Also, TNF-αlevels increased 16% in females taking the placebo with no differencesin males. Regarding the lymphocyte population, there was a significantreduction from 3.05% to 2.44% in the γ δ-T cell population with theintake of placebo regardless of gender. Regarding the number of healthydays and illness symptoms, no significant difference was found betweentime and treatments.

Conclusion: Overall, the accessed outcomes were affected withineach intervention, but there were no significant differences betweentreatments. In addition, the present study did not find a health benefiton cold/flu symptoms by taking GSH in an aged healthy cohort whencompared to placebo.

Funding SourcesKyowa Hakko Bio Co., Ltd (Tokyo, Japan).

Daily Consumption of Aronia melanocarpa (chokeberry) Pow-ders Improves Endothelial Function in Healthy Individuals: ARandomized Controlled Trial (P08-097)

Ana Rodriguez-Mateos,1 Geoffrey Istas,1 Eleonor Wood,1 Clau-dia Rawlings,1 Jeeyoung Yoon,1 Vaishnavi Dandavate,1 MelanieLe Sayec,1 Emilie Fromentin,2 andAna Rodriguez-Mateos,1

1King’s College London, UK; and 2Naturex

Objective: Aronia melanocarpa is a rich source of polyphenoliccompounds. Studies focusing on the cardiovascular health of berry(poly)phenols so far have been conducted with relatively high dosesof fruit or juice that are impractical for daily consumption. This studyaims to investigate the effect of low doses ofAronia powders on vascularfunction in young normotensive healthy males.

Methods: A double-blind, parallel, controlled trial was conductedin 66 healthy male adults randomly allocated to receive a capsuleof 500 mg of either Aronox (Aronia extract standardized to 40%polyphenols), Aronia Full Spectrum (Aronia whole-fruit powder), orplacebo (artificially coloured maltodextrin). Each participant ingested1 capsule every day for 12 wk. Flow-mediated dilation (FMD), pulsewave velocity, augmentation index, blood pressure, and heart rate wereassessed at baseline and 2 h on day 1 and 12wk later. Blood samples weretaken at the same time points for serum biochemistry and (poly)phenolanalysis.

Results: Consumption of Aronox and Aronia Full Spectrumfor 12 wk led to a significant increase in FMD compared withcontrol (DFMD: 1.1 ± 0.2% and 0.7 ± 0.2% compared with –0.1 ± 0.2%, P = 0.001 and P = 0.044, respectively). Aronox alsosignificantly increased FMD 2 h postconsumption with respect tocontrol (DFMD: 1.5 ± 0.2 compared with 0.3 ± 0.2 respectively,P = 0.02). No statistically significant differences were found for othermeasurements.

Conclusions: In healthy young men, daily consumption of Aronoxand Aronia Full Spectrum for 12 wk improved endothelial function,indicating that regular Aronia berry consumption has the potential tomaintain cardiovascular health even in healthy individuals at low riskof cardiovascular disease.

Funding SourcesNaturex.

Dietary Tomato or Lycopene Intake and the Emergence ofCastration-Resistant Prostate Cancer in the Transgenic Adenocarci-noma of the Mouse Prostate (TRAMP) Cancer Model (P08-098)

Joe L Rowles,1 Catherine Applegate,1 Rita J Miller,1 Steven KClinton,2 William D O’Brien,1 and John Erdman1

1University of Illinois at Urbana-Champaign; and 2The Ohio StateUniversity

Objective:Epidemiologic evidence suggests that tomato products orlycopene are associated with reduced prostate cancer (PCa) incidence,yet little information exists regarding the ability of tomato or itsbioactive components to impact PCa therapy. Castration-resistantprostate cancer (CRPC) is the result of progression following androgendeprivation therapy (ADT). CRPC is an advanced phase of PCa inhumans with a poor prognosis. In our present study, we hypothesizedthat dietary tomato or an equivalent concentration of lycopenefollowing orchiectomy would reduce tumor burden and progressionin transgenic adenocarcinoma of the mouse prostate (TRAMP) micecompared with a contros diet.

Methods: To test this hypothesis, male TRAMPmice (n = 90) wereprovided a powderedAIN-93Gdiet (BASE) until 12wk of age. Tomodelthe effects of ADT (reducing serum testosterone), mice were castratedat 12 wk. After castration, animals consumed either BASE with placebobeadlets (Placebo, n = 30), an AIN-93G diet modified to contain 10%lyophilized tomato paste (TP; n = 30), or BASE fed lycopene at aconcentration matched to TP (LYCO, n = 30). Prostates of TRAMPmice were monitored by ultrasound for in vivo tumor detection and3-dimensional volumetric growth measurement. After the mice werekilled, tissues were collected, and carotenoids measured by (HPLC) inthe tumor, serum, liver, and epidydimal adipose tissue. Prostate tissueand all suspected metastatic sites were harvested, processed, stained,and evaluated by histopathologic criteria.

Results: This study is the first to compare the effects of tomatopowder and lycopene on PCa progression in the TRAMP model fol-lowing castration. In a preliminary study we have observed that tomatopowder following orchiectomy (ADT) was sufficient to reduce CRPCgrowth rate and volume in this model. Accordingly, we hypothesizethat TP and LYCO will both reduce CRPC tumor recurrence andCRPC tumor growth, and extend tumor-free survival compared withPlacebo, and that TP will have a greater reduction than LYCO due tothe additional carotenoids that may accumulate in the tumor to protectagainst progression.

Funding SourcesThis work was supported by USDA NIFA ILLU-971-334 and NIH

R37EB002641.


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Amaranth Peptides Decreased the Activity and Expression ofCellular Tissue Factor on Lipopolysaccharide-Activated THP-1HumanMonocytes (P08-099)

Ana Clara Sabbione,1 Diego Luna-Vital,2 María Cristina Añón,1and Elvira Gonzalez de Mejia2

1Centro de Investigación y Desarrollo en Criotecnología de Alimen-tos (CIDCA), Argentina; and 2University of Illinois

Objectives: The aim of the present research was to evaluate theantithrombotic and anti-inflammatory effect of amaranth peptides ontissue factor (TF) activity, and the expression of THP-1–activated cells.

Methods: Amaranthus mantegazzianus protein isolates were ob-tained through alkaline precipitation, and simulated gastrointestinaldigestion (SGD) was performed with the use of pepsin and a pancreatinpool of enzymes. An active anticoagulant peptide fraction (AF)was acquired by fast protein liquid chromatography. Human THP-1 monocytes were treated for 24 h with different concentrations ofsterile-filtered SGD and AF; then cells were activated with 10 μg/mLlipopolysaccharide (LPS) dissolved in growth medium for 4 h. TFexpression was measured by Western blot and the activity wasanalyzed through a tissue factor human chromogenic activity assay.Immunocytochemical fluorescence confocal microscopy analysis afteramaranth peptide treatment was also performed. The expression profileof thrombosis-inflammation–related proteins was evaluated with aHuman XL Cytokine Array.

Results: The AF was found to inhibit TF expression (IC50 = 0.39mg/mL) and activity (IC50 = 0.58 mg/mL). Localization of TFshowed that treated monocytes decreased 49% in the membrane andincreased 2-fold in nuclei compared with positive control (P < 0.05),indicating that the treatment reduced active membrane TF anddecreased the initiation of the coagulation cascade. Moreover, thecytokine array indicated that AF peptides suppressed the expressionof MIP-3α, interleukin-1β , interleukin-1α, TARC, PDGF-AA, andPentaxin 3 compared with Ctrl+, with reductions of 79%, 62%, 54%,43%, 38%, and 38%, respectively (P < 0.05). According to theseresults, the cytokines with the highest inhibition of expression wereinvolved in the induction of the nuclear transcription factor κB (NF-κB) pathway and the platelet adhesion, aggregation, and activationprocess.

Conclusions:The results provide potentialmechanistic informationon the antithrombotic and anti-inflammatory effect of AF, indicatingthat amaranth peptides could inhibit TF expression by producinga negative feedback regulator role over the NF-κB pathway. Thiseffect could result in the prevention of hypercoagulability states andsubsequent thrombosis and inflammation pathologies.

Funding SourcesThe Fulbright Scholar Program provided research grant support.

Pharmacokinetic Parameters of Red Raspberry Polyphenols inHuman Plasma over a 24-Hour Period (P08-100)

Amandeep Sandhu, Jiayi Fan, Di Xiao, Indika Edirisinghe, andBritt Burton-Freeman

Illinois Institute of Technology

Objective: Red raspberries possess a unique polyphenol profile,mainly consisting of anthocyanins and ellagitannins. These polyphe-nols, along with various metabolites generated in the body during theprocess of digestion/absorption, are hypothesized to play a role in re-ducing disease risk. Accordingly, understanding their pharmacokinetic(PK) behavior in humans could guide intake recommendations foroptimizing their benefits. The objective of this study was to identifymetabolites generated after raspberry intake and characterize their PKprofile over 24 h.

Methods: Middle-aged overweight or obese adults (n = 28; age36 ± 13 y; body mass index 30.3 ± 5.1 kg/m2) participated in a 3-arm, randomized, single-blinded, crossover controlled clinical trial.Subjects were provided either with a control (0 g red raspberries),1 cup (125 g) frozen red raspberries or 2 cups (250 g) frozen redraspberries along with a high-fat and high-carbohydrate meal on 3separate occasions. Blood samples were collected at baseline (t = 0h)and then at the following time points: 0.5, 1, 2, 4, 6, 7, 8, and 24 h.Red raspberry polyphenols (anthocyanins and ellagitannins) and theirmetabolites (glucuronides, urolithins, phenolic acid derivatives) wereextracted from plasma at each time point and quantified by ultra-high-performance liquid chromatography coupled with triple-quadrupolemass spectrometry.

Results: Cyanidin-3-O-sophoroside was the major anthoyanindetected in plasma, with amaximum concentration of 8.3± 2.8 nmol/Lat 2 h. Urolithin A and B glucuronide appeared in the plasma at 24 hafter red raspberry intake with concentrations of 0.3± 0.2 and 0.2± 0.2nmol/L, respectively. Among phenolic acids, 4-hydroxybenzoic acidshowed a biphasic pattern peaking at ∼1 h (3.3 ± 1.3 µmol/L)and at ∼7 h (1.9 ± 0.5 µmol/L) after red raspberry intake. Theconcentrations of ferulic/isoferulic acid glucuronide, p-coumaric acid,3,4-dihydroxybenzoic acid, isohomovanillic acid, and homovanillicacid increased after red raspberry intake.

Conclusion: The results indicate that red raspberry polyphenolsare absorbed and extensively metabolized in the body, and theconcentration of various metabolites increased in a dose-dependentmanner.

Funding SourcesNational Processed Raspberry Council.

Effect of Smallanthus sonchifolius Roots on the Prevention ofGlucose and Lipid Metabolism Disorders in Mice Fed a High-Fructose Diet (P08-101)

Maria Santos,1 Eduardo Simoni,2 Karine E Silva,2 Michael EOliveira,2 Viviany S Chagas,2 and Leticia Oliveira2

1Universidade Federal do Triângulo Mineiro, Brazil; and2Universidade Federal de São João Del Rei, Brazil

Objectives: The increase in excess weight gain worldwide is closelyrelated to the occurrence of several metabolic disorders, such asinsulin resistance, type II diabetes, dyslipidemia, and fatty liver disease.A high-fructose (HF) diet might play a role in the onset of thosemetabolic disorders. The tuberous yacon [Smallanthus sonchifolius (Ss)]root is rich in bioactive substances. It contains high levels of fructo-oligosaccharides (FOS) and also some inulin and phenolic compounds.Some studies have shown that Ss has antioxidant, hypoglycemic, and


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hypolipidemic properties. For most of the studies that use Ss, theroot powder was obtained via classic extraction techniques such asmaceration followed by organic extraction and evaporation. In thisstudy, our Ss extract was obtained via a spray-drying encapsulationtechnique, which we expected would minimize the loss of bioactivity.The aim of this work was to evaluate the atomized Ss extractto prevent glucose and lipid metabolism disorders in mice fed anHF diet.

Methods: Swiss male mice (n = 6/group) were fed for 8 wkwith: balanced diet + water (C), balanced diet enriched with 5% ofSs + water (CS), balanced diet + 30% of fructose in water (HF),and balanced diet enriched with 5% of Ss + 30% of fructose inwater (HFS). Food (g) and water (mL) were measured daily. Bodyweight (g)wasmeasuredweekly. After 8wk, biochemicalmeasurements(mg/dL), including glucose (GLU), total cholesterol (CHO), high-density lipoprotein (HDL), and tryglicerides (TGs), were evaluatedin blood through the use of commercial kits. Liver lipid content,liver histology, and glucose tolerance test (GTT) were also performed.Results were expressed as mean ± SEM. Data were analyzed by t test;P < 0.05 was considered significant for all data.

Results:HFmice showed an increase in adipose tissue depots, GLU,CTO, HDL, and liver lipid content, and an impaired GTT and liverhistology. On the other hand, HFS mice showed a decrease in liver lipidcontent, GLU, and also an improvement in GTT, liver lipid content, andhistology.

Conclusion: It seems that Ss was able to attenuate glucose and lipidmetabolism alterations in mice fed an HF diet. It is possible that morepronounced results could be achieved by increasing the dose and alsothe period of Ss administration.

Funding SourcesCNPq.

Lycopene Attenuates High-Fat-Diet–Induced Oxidative Stress inSprague-Dawley Rats (P08-102)

Katelyn E Senkus, Libo Tan, and Kristi M Crowe-White

The University of Alabama

Objective: Dietary patterns high in fat promote adipose tissue (AT)accumulation, leading to production of free radicals and overt oxidativestress. The purpose of this study was to evaluate the influence oflycopene, a lipophilic carotenoid with antioxidant functionality, onantioxidant-oxidant balance in Sprague-Dawley rats fed diets meetingand exceeding recommendations for fat intake in humans.

Methods: Male Sprague-Dawley rats (n = 18) aged 4 wk were fed30% fat or 60% fat purified diet supplemented with 100 mg lycopene/d.Three rats in each diet group were killed at weeks 3, 7, and 10 toassess durational effects of lycopene supplementation on antioxidant-oxidant balance mediated by dietary fat and AT accumulation. Dailyfood intake was recorded as was body weight and visceral ATweight when the animals were killed. Serum lipid peroxides wereassayed by thiobarbituric acid-reactive substances (TBARS), and serumantioxidant capacity (AC) was assessed based on the oxygen radicalabsorbance capacity assay.

Results:At 3 wk, TBARSwere significantly inversely associated withlycopene intake (P = 0.039, r = –0.897). In addition, TBARS were

significantly higher in rats consuming 60% fat diet (P = 0.024). At 7wk, lycopene intake was significantly higher among rats consuming30% fat diet, yet no significant differences in TBARS, AC, or ATaccumulation were observed between groups. At 10 wk, nonsignificantdifferences in lycopene intake or TBARS were noted; however, AT andlipophilic AC were significantly greater among rats consuming 60%fat diet (P = 0.028 and P = 0.042, respectively). Lycopene intake wassignificantly predictive of TBARS (P = 0.007) only through 3 wk ofsupplementation.

Conclusions: Significant increases in AT were observed among ratsfed 60% fat diet for 10 wk concurrently with nonsignificant differencesin body weight and oxidative stress between diet groups. The resultssupport the hypothesis that lycopene attenuates high-fat-diet–inducedoxidative stress.

Funding SourcesNA.

Intake ofWhole Fruit, Polyphenol-Rich and Fiber-Rich Fractionsof Red Raspberry on Food Efficiency and Glucose Regulation inHigh-Fat-Fed Mice (P08-103)

Neil F Shay,Marlena Sturm,Alexandra Becraft, andRufaMendez

Oregon State University

Objectives: Consumption of red raspberries has been shown toproduce beneficial effects on metabolism, presumably due to theirrelatively low sugar content, and high fiber and high phenolic content.Previous studies suggest whole raspberry consumption remediatesweight gain and induces a healthfulmetabolic shift in high-fat-fedmice.We tested here the effect of the following: 1) whole red rasberry pureeconcentrate (RPC); 2) a polyphenol-rich fraction (RP), and 3) a fiber-rich fraction (RF) in mice fed an unhealthy high-fat, Western-stylediet.

Methods: Raspberry puree concentrate was used to produce 2 foodfractions: RP and RF. These fractions were obtained via amberlite affin-ity hydrophobic chromatography and enzymatic methods, respectively.The whole food RPC and RP and RF fractions were fed for 10 wk toC57BL/6J male mice fed a high-fat, high-cholesterol, and high-sucrose(HF) diet. A second control group was fed a low-fat (LF) diet. Bodyweight and food intake was measured weekly, and glucose tolerance testand insulin sensitivity test were administered in weeks 8 and 9. After10 wk, mice were killed and serum and tissues collected for furtheranalysis.

Results: Intake of RPC,RP, andRFwith theHFdiet improved fastingglucose levels such that values were statistically indistinguishable frommice fed the healthy LF diet. Glucose tolerance testing indicated that thearea under the curve was improved with RPC consumption (P < 0.05)and trended to significance (P < 0.10) with intake of both RP and RFwith HF. Weight gain showed no significant differences in all HF-fedmice; however, food efficiency was favorably affected in HF-fed groupsprovided with both RPC and RP (P < 0.05)

Conclusions: As shown previously in 2 different studies, the intakeof RPC along with an HF diet improves glucose metabolism comparedwith mice fed HF alone. Fasting blood glucose levels were improvedin the RPC, RP, and RF groups compared with the HF-fed mice, andfood efficiency was improved in mice fed RPC and RP compared with


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HF-fed mice. In an effort to determine whether RP or RF is the mainfraction responsible for the beneficial effect of RPC, the present resultsare not conclusive, in part because it is likely that both RP and RF arecontributing to the health and metabolic improvements seen in HF-fedmice fed RPC.

Funding SourcesNational Processed Raspberry Council.

Intake of Watermelon and Watermelon Byproducts ImproveGlucoseMetabolismandFoodEfficiency inHigh-Fat-FedMice (P08-104)

Neil F Shay,Alexandra Becraft,Marlena Sturm, andRufaMendez

Oregon State University

Objectives:Watermelon is a unique and nutritious fruit containingphytochemicals that have a beneficial effect on metabolism when con-sumed. In particular, a strong body of evidence demonstrates benefitsof watermelon intake on blood pressure regulation along with otherhealth benefits. In the present study, we tested the hypothesis that intakeof whole watermelon, and potential value-added products made fromwatermelon rind and skin, would remediate metabolic complicationsin C57BL/6J male mice fed a high-fat, high-cholesterol, and high-sucrose diet modeling an obesigenic and diabetigenic Western-stylediet.

Methods: Groups of mice (n = 8) were provided either low-fatdiet (LF, 10% kcal fat), high-fat diet (HF, 45% kcal fat), or HF plusWatermelon Skin (HF + WS), HF plus Watermelon Rind (HF + WR),or HF plus Watermelon Flesh (HF + WF) for 10 wk. Watermelon fleshwas provided at 10% of total energy and skin and rind were addedat ∼2% (wt/wt, dry weight of additives) of diet. Animals consumedexperimental diets ad libitum; body weight and food intake wererecorded weekly. In week 8, a glucose tolerance test was conducted,and in week 9, an insulin sensitivity test was performed. After 10 wk,animals were killed, serum collected, and liver tissue saved for RNAisolation.

Results: Fasting glucose concentrations in week 8 for the HF + WSand HF + WR groups were reduced compared with the HF-fed micesuch that they were statistically indistinguishable from the LF-fedcontrol group.A glucose tolerance test demonstrated that the area underthe curve (AUC) for the HF + WR group was reduced and again,statistically indistinguishable from the LF-fed control group. Althoughno test diet reduced body weight gain compared with HF-fed mice,food efficiency was improved in the HF + WR and HF + WS groupscompared with the HF-fedmice; their food efficiencies were statisticallyindistinguishable from the LF-fed mice.

Conclusions: At a modest level of supplementation (2% wt/wt)to HF diet, additives made from watermelon rind and skin im-proved fasting blood glucose concentrations and food efficiency;these values became statistically indistinguishable from healthy LF-fed mice. The same observation was made for glucose AUC inmice fed HF + WR. We hypothesize that WR and WS containbioactive fiber and perhaps other phytochemicals that beneficially affectmetabolism.

Funding SourcesNational Watermelon Promotion Board.

Supplementation of Curcumin Increases Lifespan in Drosophilaunder Heat Stress Conditions (P08–105)

Lirong Shen,1 Yong Chen,1 Xin Liu,1 and Chao-Qiang Lai2

1ZhejiangUniversity, China; and 2USDAARSNutritional GenomicsLaboratory, JM-USDA Human Nutrition Research Center on Aging atTufts University, MA

Objectives: Our previous work indicated that curcumin (CUR)could increase survivorship in response to heat stress. This aim of thisstudy was to examine CUR’s effect on survival of fruit flies under heatstress (HS).

Methods: Newly emerged Canton-S flies were collected under lightanesthesia and housed separately by sex. Then flies were fed 2 diets:basal diet (N) and diet with addition of 0.2 mg/g curcumin (C),respectively. These flies were treated at 3 temperatures to assess HS: 1)25°C (25) and 2) 29°C (29) for their lifespan, and 3) 34°C for 2 h (34)at days 1, 4, and 7. In total, there were 6 treatments (N25, N29, N34, C25,C29, C34) for male and female flies, with each treatment containing 300female flies or male flies in 10 vials. Flies were counted for survival andtransferred to fresh media every 2 d. Lifespan analysis was performedon pooled interval-censored data. Flies were sampled at days 7 and 21for measuring malonaldehyde level, superoxide dismutase (SOD) andcatalase (CAT) activity, and gene expression by reverse transcriptase-polymerase chain reaction.

Results: In the basal diet, compared with N25, the mean lifespan ofN29 and N34 were decreased significantly, ranging from 8.5% to 15.7%in males, and from 3.7% to 7.9% in females, indicating that HS resultedin lifespan reduction, with male flies being more vulnerable to HS. Onthe other hand, in the CUR diet, the mean lifespan of C29 and C34

were extended substantially, in the range of 8.7–16.4% in males and8.9–12.8% in females, compared to that of their corresponding basaldiet N29 and N34. Our results demonstrated that supplemental CURenhanced the survivorship and resistance to HS of the flies. In addition,supplemental CUR also palliated increased oxidative stress caused byHS, upregulated the expression of sod1 and cat, and downregulated theexpression of hsp70 and hsp83.

Conclusions: Our results confirm that CUR supplementationincreases survivorship through enhancing resistance to and alleviatingoxidation stress caused by HS.

Funding SourcesThe National Key Research and Development Program of China

(2017YFC1601700), National Natural Science Foundation of China(31271848), Foundation of Fuli Institute of Food Science of ZhejiangUniversity (KY201404), Foundation from the USDA, AgricultureResearch Service (58-1950-9-001).

Postprandial Glycemic and Insulinemic Responses toPolyphenol-Fortified High-Carbohydrate Snack Bars (P08-106)

Tracey J Smith,1 Marques Wilson,1 J Philip Karl,1 Claire CWhitney,1 Ann Barrett,2 Nicole Farhadi,2 and Scott J Montain1

1US Army Research Institute of Environmental Medicine; and2Natick Soldier Research, Development, and Engineering Center


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Background: Certain berries, when consumed whole or as nectars,appear to promote glycemic control, an effect attributed to theirpolyphenol makeup.

Objective: The aim of this study was to determine whetherpolyphenol-rich freeze-dried fruit or fruit extracts added tocarbohydrate-based foods produce a dose-dependent moderation ofthe postprandial glycemic, insulinemic, and glucoregulatory hormoneresponses.

Methods: In this randomized, crossover, placebo-controlled trial,20 participants (18 male/2 female; age 24 ± 5 y; body mass index27 ± 3 kg/m2) consumed 1 of 5 snack bars (∼88% energy fromcarbohydrate) containing no fruit ingredients (control), two levels offreeze-dried black raspberries (10% or 20% total weight; LO-R andHI-R, respectively), and two levels of cranberry extract (0.5% or 1%total weight; LO-C and HI-C), on trials separated by ≥5 d. Circulatingglucose, insulin, C-peptide, and glucose-dependent insulinotropicpolypeptide (GIP) concentrations were measured preprandial andfor 3 h postprandial. Dose-dependent effects of the raspberries andcranberries relative to control were examined with the use of separatelinear mixed models.

Results: Relative to control, HI-R resulted in 5% lower peak insulin(P = 0.03), 3% lower C-peptide (0–60 min incremental area underthe curve, AUCi; P = 0.01) and 3% lower GIP (0–120 and 0–180 minAUCi, pi or peak). LO-R did not impact glucose or hormone responsesrelative to control, but glucose AUCi (0–180 min) trended towardsbeing higher (43%) after HI-R compared with LO-R (P = 0.06). Nosignificant treatment effects were observed for the cranberry bars.

Conclusion: HI-R produced a more favorable postprandial glu-coregulatory response than the same bar fortified with no or 10% blackraspberry powder, whereas cranberry extract fortification had no effecton glycemic and insulinemic responses. Polyphenol fortification maybe a method for moderating the glycemic properties of certain foods,but its effectiveness appears dependent on polyphenol source/form anddose.

Funding SourcesUS Army Medical Research and Materiel Command.

Oxidative Stress Response to Polyphenol-Fortified Snack Bars(P08-107)

Tracey J Smith,1 James P Karl,1 Marques A Wilson,1 Ann HBarrett,2 Claire CWhitney,1 Nicole F Farhadi,2 Scott J Montain,1and C-Y Oliver Chen3

1US Army Research Institute of Environmental Medicine; 2NatickSoldier Research, Development and Engineering Center; and 3JeanMayer USDA Human Nutrition Research Center on Aging

Background: High-carbohydrate meals produce postprandiallipemia, which is associated with increased oxidative stress, acontributor to chronic diseases. Polyphenols, a heterogeneous group ofphytochemicals, are ubiquitous in plant-based foods and may protectagainst oxidative stress.

Objectives: The aim of this study was to determine whetherpolyphenol-rich freeze-dried fruit or fruit extract added to acarbohydrate-based food dose dependently reduces the postprandialoxidative stress response.

Methods: In this randomized, crossover, placebo-controlled trial, 20participants (18male, 2 female; 24± 5 y; bodymass index 27± 3 kg/m2)consumed 1 of 5 snack bars (∼100 g carbohydrates,∼460 kcal) contain-ing no fruit ingredients (control), 2 concentrations of freeze-dried blackraspberries (10 and 20% total wt), and 2 concentrations of cranberryextract (0.5 and 1% total wt) on trials separated by≥5 d. The capacity offruit polyphenols tomodulate the resistance of lipids within low-densitylipoprotein (LDL to oxidation was measured preprandially and for 3 hpostprandially by an ex vivo Cu2+-induced LDL oxidation assay. Dose-dependent effects of the black raspberries and cranberries relative tocontrol were examined with the use of separate linear mixed models.

Results: There was an overall time effect (P< 0.01), wherein the lagtime was 4–6% shorter, indicating more rapid oxidation, 180 min afterconsuming the bars (mean ± SD 121 ± 22 s) compared with the lagtime at baseline, 60 min and 120 min (P

Conclusion: At the doses provided, neither freeze-dried blackraspberries nor cranberry extract enhanced the resistance of LDLlipids to oxidation following consumption of a high-carbohydratesnack bar. Future research should determine whether the addition ofblack raspberry or cranberry polyphenol to high-fat, rather than high-carbohydrate, food items modulates resistance of LDL to oxidation.

Funding SourcesUS Army Medical Research and Materiel Command.

Administration of Purified Cyanidin-3-Glucose or a BlackberryExtract Causes Improved Mitochondrial Function but ReducedContent in 3T3-L1 Adipocytes (P08-108)

Patrick Solverson, George Albaugh, Dawn J Harrison, Dave LLuthria, David J Baer, and Janet Novotny

United States Department of Agriculture, Beltsville Human Nutri-tion Research Center

Objective: Consumption of berries elicits antiobesity and antidi-abetic effects in both animal and human studies. The application ofanthocyanin or berry extracts to adipocytes in vitro leads to increasesin insulin sensitivity, reductions in lipid droplet formation, and a white-to-brown (beige) phenotypic switch of the tissue. The objective of thisstudy was to investigate the effect of cyanidin-3-glucoside (C3G) orblackberry extract (BBE) on respiration in mature adipocytes.

Methods: In two separate experiments, 3T3-L1 adipocyte cells weretreated with 0, 150 nM, or 50 µM of purified C3G or equivalentsprovided by a BBE. The BBE experiment also included a 25 µM dose.Baseline, state 4, state 3, and uncoupled respiration states were createdexperimentally andmeasured by oxygraph. Respiration rate within eachexperimental state was corrected for mitochondrial content, which wasdetermined by PCR.

Results: Mitochondrial content was significantly reduced with thehigh dose of C3G (12.9 compared with 14.1 compared with 7.62∗2�Ct, control compared with 150 nM compared with 25 µM C3G,respectively; P = 0.007) as well as with the two higher doses of BBE(14.5 compared with 14.3 compared with 5.5 compared with 4.6 2∗2�Ct,control compared with 150 nM compared with 25 µM compared with50 µM, respectively; P = 0.0001). After correction for mitochondrialcontent, per million cells, the 50 µM dose of C3G resulted in thehighest respiration rates (control comparedwith 150nMcomparedwith


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50mM, respectively) for baseline state (7.3 comparedwith 5.1 comparedwith 10.0 pmol O2/s; P = 0.0002), state 4 (5.4 compared with 4.0compared with 7.1 pmol O2/s, P= 0.0021), state 3 (38.2 compared with27.7 compared with 56.1 pmol O2/s; P = 0.0013), and uncoupled state(79.1 compared with 57.8 compared with 109.4 pmol O2/s; P= 0.0037).The intermediate-high dose (25 mM) BBE also increased respiration(control compared with 150 nM compared with 25 mM comparedwith 50 mM) for baseline state (5.7 compared with 6.0 compared with14.2 compared with 7.8 pmol O2/s, p2/s, P = 0.0002), state 3 (29.0comparedwith 32.0 comparedwith 60.5 comparedwith 31.0 pmolO2/s,P = 0.0025), and uncoupled state (58.6 compared with 62.0 comparedwith 89.4 compared with 48.2 pmol O2/s, P = 0.0107).

Conclusion: Administration of a purified anthocyanin or a berryextract caused decreases in mitochondrial content, but increases inmitochondrial function in cultured adipocytes. These observationssupport the antiobesity effects associated with anthocyanins noted inearlier studies.

Funding SourcesUS Department of Agriculture.

Effect of Extraction Solvent and Method on the AntioxidativeActivities ofMoringa oleifera Lam. Leaves (P08-109)

Mi Jeong Song and Hyun-Sook Kim

Sookmyung Women’s University, South Korea

Objective:Moringa oleiferaLam. (MO) is recognized as an abundantsource of phytochemicals and has been reported to exert multiplebiologic effects, including antioxidant, anti-inflammatory, antidiabetic,and anticancer effects. Among the parts of the plant, the leaves of MOin particular contain significant amounts of phenolic and flavonoid

FIGURE P08-109-1

compounds that can be obtained by water or ethanol extraction. In thisstudy, we investigated whether different extraction methods will affectthe antioxidative activity and phytochemical compounds of ethanolextract from the leaves of MO.

Methods: Dried powdered MO leaf samples were divided into4 groups and subjected to different extraction methods: raw groupthat received no treatment (R); water extraction (W); stirred ethanolextraction (ES); refluxed ethanol extraction (ER) (Figure 1). For theanalysis, we extracted 4 samples in methanol solution. The DPPHand ABTS radical scavenging activity was assessed with 1 mM DPPHmethanol solution and 2.5 mM ABTS solution that were addedimmediately prior to the experiment. The total phenolic compoundcontent was calculated on a gallic acid standard curve and the totalflavonoid content was based on a quercetin standard curve (Figure 2).

Results: The DPPH radical scavenging activity was the highest inW(89.06%) and lowest in ES (28.98%) among the sample groups, but allgroups were significantly lower than ascorbic acid (92.20%) (Figure 3).The ABTS radical scavenging activity of R (94.12%) and W (94.32%)was higher than ER (93.51%) (Figure 4). The total phenolic compoundswere 62.4 mg GAE/100 g (R), 126.27 mg GAE/100 g (W), 265.20mg GAE/100 g (ES), and 286.80 mg GAE/g (ER). The total flavonoidcompounds were 266.0 mg QE/100 g (R), 360.0 mg QE/100 g (W),1546.67 mg QE/100 g (ES) and 1353.33 mg QE/100 g (ER).

Conclusions: Our results suggest that the antioxidant abilities, andthe phenolic and flavonoid compound contents of MO extracts wereaffected by different extract solvents and methods.

Funding SourcesThere are no funding sources for this research.



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FIGURE P08-109-2

FIGURE P08-109-3


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FIGURE P08-109-4

Metabolic Effects of Dietary Supplementation of Lycium bar-barum Polysaccharides on Serum and Urine Metabolomics amonga Young Healthy Male Population (P08-110)

Guiju Sun and Hui Xia

Key Laboratory of Environmental Medicine and Engineering ofMinistry of Education, andDepartment ofNutrition and FoodHygiene,School of Public Health, Southeast University, China

Objective: The aim of this study was to observe the metabolomicseffects of Lycium barbarum polysaccharides (LBPs) on young healthymen.

Methods: High-performance gel permeation chromatography andion chromatography were used to detect LBP composition. Forty-twovolunteers were divided into 2 groups (n = 21 each) and required totake 2 capsules every day (300 mg/capsule) for 4 wk to explore changesin anthropometric, biochemical indexes, and metabolic profiles.

Results: LBP, with a molecular weight of 3.74 kDa, consisted offucose, rhamnose, aminogalactose, galactose, glucose, mannose, andfructose in a molar ratio of 0.02:0.08:0.03:0.11:46.67:0.37:4.72. Afterscreening, d-talose decreased, phosphate and leucine increased sig-nificantly in serum. 4-Hydroxymandelonitrile increased, but tyrosine-1,3-aminoisobutyric acid, threitol and ribose decreased significantly inurine. Following pathway searching, phenylalanine, tyrosine and tryp-tophan biosynthesis, and tyrosine and glycerophospholipidmetabolismwere strongly related to the metabolism of the LBP supplement.

Conclusion: Eight metabolites were simultaneously assessed afterLBP supplementation inmales. LBPmay influence the glycerophospho-lipid metabolism and tyrosine metabolism.

Funding SourcesNational Key Research and Development Program of China

(2016YFD0400604-02), the National Natural Science Foundation ofChina (81273069), and the Fundamental Research Funds for theCentralUniversities (2242016K40024).

Interorgan β-Hydroxy-β-Methylbutyric Acid (HMB) andBranched-Chain Keto Acid (BCKA) Transport in the HealthyPig (P08-111)

Gabriella A Ten Have, Lisa Jansen, Marielle Engelen, andNicolaas Deutz


Objective: Branched-chain amino acids (BCAAs; leu + ile + val)and their metabolites the branched-chain keto acids [BCKAs;α-ketoisocaproic acid (kic) + α-keto-β-methyl-n-valeric acid(kmv) + 2-oxoisovalerate (kiv)] and the kic metabolite β-hydroxy-β-methylbutyric acid (HMB) are involved in the regulation of keysignaling pathways in the anabolic response to a meal. However, their(inter)organ production and transport rates are unknown. Therefore,we studied BCAA, BCKA, and HMB kinetics by measuring theiracross-organ net balances in the postabsorptive and postprandial statein a conscious pig model.

Methods: In multicatheterized pigs (n = 12, ± 25 kg), net balancesacross liver, portal-drained viscera (PDV), kidney, and hindquarter(HQ, muscle compartment) were measured before and up to 4 h afterbolus feeding of a completemeal (30%daily intake). Arterial and venousplasmawere collected and concentrationsweremeasured by LC- orGC-MS/MS.Data expressed asmean± SE. Significance (P< 0.05) fromzerowas assessed by Wilcoxon Signed Rank test.

Results: The systemic availability time courses of the individualBCKAs parallel their BCAAs but that of HMB was more delayedand prolonged (Tmax leu and kic: 60 min; HMB: 180 min). In thepostabsorptive state, HMB was significantly taken up by the kidney(3.2 ± 0.8 µmol · kg body wt–1 · min–1; P = 0.001) but no significantBCKA production or uptake was observed. In the postprandial state,the total net balances over 4 h (mmol · kg body wt–1 · 4 h–1) showedsignificant net production of BCKA in HQ (kic: 25.2 ± 2.2, P = 0.001;kmv: 10.2 ± 2.7, P = 0.004; kiv: 10.8 ± 1.2, P = 0.001), kic in PDV


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(12.3 ± 2.4, P = 0.002), and kiv in kidney (10.0 ± 3.4, P = 0.01). HMBwas only produced in the liver (0.76 ± 0.11; P = 0.0005). All BCKAswere significantly taken up by the liver (kic: 123 ± 19, P = 0.004; kmv:34.6 ± 6.5, P = 0.0007; kiv: 41.1 ± 4.7, P = 0.004) and HMB was takenup by the kidney (0.98 ± 0.20; P = 0.0008).

Conclusions: Substantial differences are present in (inter)organmetabolism and transport among the BCAAs and their metabolitesBCKAs and HMB. We hypothesize that this is related to differences inthe regulation of key signaling pathways in the anabolic response to ameal in health and disease.

Screening of Potential Compounds to Enhance the Anti-Inflammatory Effect of Palmitoylethanolamide (P08-112)

Ariela B Thomas, Susanne Mertens-Talcott, Vinicius de PaulaVenancio, and Stephen Talcott


Objectives: Palmitoylethanolamide (PEA) is an endogenous fattyacid amide belonging to the N-acylethanolamine (NAE) family. Previ-ous studies have demonstrated anti-inflammatory activities of PEA invivo and in vitro.We hypothesized that the anti-inflammatory activitiesof PEA could be enhanced in combination with bioactive plant extractsfrom Boswellia serrata (Bosw) and capsaicinoids (Caps), which havebeen described to decrease inflammation. The objective of this studywas to evaluate Bosw and Caps as potential anti-inflammatory agentsin combination with PEA.

Methods: Murine macrophages (RAW264.7) were pretreated withPEA, Bosw, and Caps at 2.5, 5.0, and 12 mg/L, respectively, for1 h, followed by lipopolysaccharide (LPS) (10 ng/mL) to induceinflammation over 8 h. The gene expression of inflammatory markersinterleukin 1β (Il-1β) and cyclooxygenase 2 (Cox-2) was evaluated byquantitative real-time polymerase chain reaction.

Results: PEA and Bosw extract alone significantly reduced Il-1βmRNA expression to 0.36- and 0.66-fold, respectively. Both combi-nations reduced mRNA expression of Il-1β , although the combinedcompounds did not present a significantly enhanced anti-inflammatoryeffect compared with PEA treatment alone.

Conclusions: The results confirm the anti-inflammatory activity ofPEA, Bosw, and Caps individually and in combination.

Telomerase Activator 65 (TA-65) Favorably Affects Parameters ofMetabolic Syndrome. A Pilot Study (P08–113)

Minu Sara Thomas, Bruno Lemos, Diana DiMarco, MelissaMelough, Amanda Missimer, Gabriela Murillo, Ock K Chun,Hana Alyousef, Isabel Medina-Vera, Maria-Luz Fernandez, andOck Chun


Background: Telomerase Activator 65 (TA-65), a compound ex-tracted from Astragalus membranaceus, was developed to increase ormaintain telomere length.

Objective: The aim of this study was to determine the effects of TA-65 on the parameters of metabolic syndrome (MetS)

Methods: We recruited 40 patients aged 32–70 y with MetS todetermine the effects of TA-65 on dyslipidemias and anthropometrics in

this at-risk population. This was a double-blind, randomized crossoverdesign inwhich patients were allocated to consume either 16mg daily ofa TA-65 supplement or a placebo for 12 wk. Following a 3-wk washout,participants were allocated to the alternate treatment for an additional12 wk. Anthropometric and biological markers were measured at theend of each treatment. Plasma lipids, glucose, C-reactive protein (CRP),liver enzymes, and glycosylated hemoglobin were measured with aCobas c-111. Plasma insulin was measured by Luminex technology andtelomere length was measured in lymphocytes and granulocytes, Dietrecords were analyzed through the use of the Nutrition Data System forResearch (NDSR).

Results: There were no changes in dietary intake between periodsand most of the parameters of MetS were not altered. Comparedto the placebo period, high-density lipoprotein (HDL) cholesterolwas higher, whereas body mass index, waist circumference, and thelow-density lipoprotein (LDL)/HDL ratio were lower (P < 0.05)during TA-65 treatment. Positive correlations were observed inchanges between the placebo and the TA-65 periods in HDLcholesterol and CRP (r = −0.511, P < 0.01) and HDL cholesteroland alanine aminotransferase (r = –0.61, P < 0.001) suggesting thatthe favorable changes observed in HDL were associated with decreasesin inflammation. Telomere length was not changed from baseline evenafter 6 mo of intervention.

Conclusion: TA-65 improved key markers of cardiovascular diseaserisk, and maintained telomere length in patients with MetS.

Funding SourcesTA Sciences.

Effect of Pueraria tuberosa on Experimental Models of ChronicDisease, i.e., Diabetic Nephropathy (P08-114)

Yamini Bhusan Tripathi and Rashmi Shukla

Banaras Hindu University, India

Objective:Multiple causative factors are involved in the mechanismof diabetic nephropathy (DN), and existing therapies do not provideperfectmedication forDNpatients.Medicinal plants and their bioactiveconstituents may prove to be safer and more effective against variouschronic diseases such as DN. This study was designed to investigate theprotective effect of Pueraria tuberosa, a traditional Ayurveda medicineused for antiaging, on DN in a rat model, and to explore the possiblemechanism involved behind its nephroprotective function.

Methods: The chemical compositions of an aqueous extract of P.tuberosa (PTY-2r) were analyzed by GC-MS. STZ (55 mg/kg bodyweight, intraperitoneal)–induced rats were randomly divided into 3groups. PTY-2r was orally given to rats for 20 d. Age-matched normalrats served as normal control (NC). Reactive oxygen species (ROS), lipidperoxidation (LPO), and the activities of ROS-scavenging enzymes,namely superoxide dismutase (SOD), catalase (CAT), and glutathioneperoxidase (GPx), were determined in kidney tissue. The expression ofapoptosis-related proteinswasmeasured through immunofluorescence.

Results: GC-MS analysis of PTY-2r indicated the presence of 37compounds, of which 4 were found in higher amounts. A significantincrease in ROS and LPO was observed along with decreased activityof antioxidant enzymes, which is responsible for oxidative stress inthe kidney of DN rats. High oxidative stress induces apoptosis in


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target cells, indicated by the significantly decreased expression ofBcl-2 along with increased expression of Bax, active Caspase-3 andcleaved PARP-1 in DN control rats. The PTY-2r treatment significantlyraised the activity of antioxidant enzymes and suppressed apoptosis,thus preventing urinary albumin excretion in a dose-dependentmanner.

Conclusion: These findings suggest that PTY-2r has nephroprotec-tive potential against STZ-induced DN rats via suppression of oxidativestress-induced apoptosis due to the presence of different bioactivecompounds.

Combined Neuroprotective Effect of Melatonin, ProtocatechuicAcid andHydroxytyrosol onα-SynucleinFibril Formation (P08-115)

Ana M Troncoso, Ruth Hornedo-Ortega, Marta Gallardo-Fernandez, Ana Belen Cerezo, and Mª Carmen Garcia-Parrilla

Universidad de Sevilla, Spain

Objective: Abnormal aggregates of α-synuclein (αsyn) in the brainare considered a relevant physiopathologic feature of Parkinson’sdisease (PD). Food bioactive compounds are being intensivelyscrutinized as potential preventive agents against PD. Recent researchhas demonstrated a potent effect against αsyn aggregation and toxicityby melatonin (MEL), protocatechuic acid (PCA), and hydroxytyrosol(HT), which are present in a variety of plant foods. Hence, followingthe consumption of a balanced diet, significant plasma concentrationsof these compounds can be achieved. The hypothesis of this study isthat if these molecules have an individual inhibitory effect on αsynaggregation, the combination of them might enhance the neuroprotec-tive effect. Therefore, the objective of this work was to evaluate the invitro inhibitory effects of MEL, PCA, and HT and their combinationson αsyn fibril formation in order to ascertain their combined dietaryeffect.

Methods: αsyn fibril formation was monitored via Thioflavin T(ThT). MEL (250 μM), PCA (100 μM), HT (100 μM), MEL + PCA,MEL + HT, PCA + HT, and MEL + PCA + HT were incubatedwith or without αsyn protein (70 µM) and ThT (25 µM) for 142 hunder continuous stirring, at 37°C and 1000 rpm. Fluorescence datawere recorded every 2 h. For ThT assay, the resulting samples wereplaced on carbon-coated Formvar grids, incubated, and viewed with atransmission electron microscope (80 kV). ThT samples were dilutedwith loading buffer and boiled at 50°C for 3 min. They were then wereloaded on 4−20% Tris-glycine gel and finally stained with coomassieblue.

Results: As can be observed in Figure 1 and Table 1, MEL, PCA,and HT (250, 100, and 100 µM, respectively) each have an inhibitoryeffect alone on αsyn fibril formation, presenting inhibition percentagesof 8.5%, 76.3% and 80.9%, respectively. MEL shows a negligible effecteither isolated or combined. The best results are obtained with acombination of the polyphenols PCA + HT (87.5%). Transmissionelectron microscopy and electrophoresis images confirm the obtainedresults (Figure 2).

Conclusion:This work has demonstrated a discrete combined effectmostly for PCA and HT, which could be achieved with an intake ofvirgin olive oil and red fruits. Nuclear magnetic resonance studies arenecessary to explain the structure-activity relation that underlies theseresults.

Funding SourcesSpanish government for its financial assistance (projects MICINN

AGL2013-47300-C3-2-R and MICINN AGL2016-77505-C3-2-R).

Grapefruit Juice and Intestinal Drug Metabolism (P08-116)

Margit E Trotz,1 Diana Varela-Margolles,2 and Jane Rugh3

1St George’s University, St George’s True Blue, West Indies; 2SGUMedical School, Grenada; and 3Self-employed

Objective: Grapefruit juice (GFJ) ingestion improves human healthbut should be avoided with certain medication. This well-known fact isoften difficult to explain to patients or students, especially since thereis the misconception that drug metabolism would mainly be affected inthe hepatocytes. The objective of this study is to interrelate publisheddata and to generate a simplified graph that clarifies how GFJ interfereswith drugmetabolism in human enterocytes, and explains possible toxiceffects when the oral drug dosage is not lowered.

Methods:Clincal studies or experiments that used human intestinalcells are included. Different types of GFJ are studied, along withtheir purified bioactive compounds, i.e., flavonoids and furanocour-marins. Results regarding GFJ and concomitant drug metabolismby cytochromes P450 (CYP) are combined with the data obtainedwhen GFJ interferes with drug transport by apical plasma membranetransporters.

Results: The CYP profile in the human small intestine shows adifferent profile to that of the liver and contains mainly CYP3A.CYP activities related to specific drug degradation are inhibited afteringestion of 1 glass of GFJ. The timeline shows a bioactive effect after4 h that lasts ≥24 h. At the same time GFJ reduces the activity ofthe enterocyte efflux transporter P glycoprotein, which is an ABC-transporter that actively pumps lipophilic molecules back into theintestinal lumen. The inhibition of CYP degradation of specific drugsand the reduction of their release back into the intestinal lumen leads toan increased area under the plasma concentration-time curve (AUC) byGFJ. Other intestinal transporters, such as organic anion-transportingpolypeptide, are currently being studied regarding the role they playin the interference of drug transport by GFJ and many other fruitjuices.

Conclusion: The inhibited presystemic drug metabolism occurringin the enterocytes explains how GFJ leads to increased AUC of specificdrugs. It also indicates that coadministration of GFJ with certain drugscan be beneficial in two respects: increased bioavailability at lower oraldosages, as well as the many beneficial health effects of GFJ.

Funding SourcesThere was no funding for this research.

Red Raspberry (Rubus idaeus) Consumption Restores the Im-paired Phenylephrine-Induced Vasoconstriction in the Aorta of theObese Zucker rat, a Model of Metabolic Syndrome (P08-117)

Natalie VandenAkker, Stefano Vendrame, PanagiotisTsakiroglou, and Dorothy Klimis-Zacas

University of Maine

Objective: Metabolic syndrome (MetS) is associated with anincreased risk of cardiovascular disease (CVD). We investigated the


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effect of whole red raspberry (WRR) consumption on vascular function(vasoconstriction) in the obese Zucker rat (OZR), a model of MetS.

Methods: At 8 wk of age, male OZRs (n = 16) and their leanlittermates (LZR) (n = 16) were placed either on a control (C)or an 8% wt/wt WRR-enriched diet for 8 wk. Aortic rings wereisolated and phenylephrine (Phe)-mediated vasoconstriction in theabsence or presence of specific inhibitors l-N-monomethyl-arginine(l-NMMA) and mefenamic acid (MFA) to determine the contributionof nitric oxide synthase (NOS) and cyclooxygenases (COX) pathways,respectively, was examined. The maximal force (Fmax) of contrac-tion and vessel sensitivity (pD2) under the above conditions weremeasured.

Results: Phe-induced vasoconstriction was lower in the OZR-C(Fmax 0.75 ± 0.05 g, P < 0.05) compared with LZR-C (Fmax 1.15 ±0.05 g, P < 0.05). The WRR diet partially restored the impairedaortic vessel vasoconstriction in the OZR aorta (Fmax 0.98 ± 0.03g, P < 0.05) compared with the OZR-C group (Fmax 0.75 ± 0.03 g,P < 0.05). When aortic rings were pretreated with the NOS inhibitorl-NMMA or the COX inhibitor MFA, the constrictor response toPhe increased in the OZR group only. The vasoconstrictor responseof the OZR-WRR group treated with l-NMMA increased comparedwith the C group (Fmax 2.03 ± 0.09 g compared with 1.82 ± 0.09 g,respectively; P < 0.05), elevating the response level to that of the LZR.Pretreatment with MFA resulted in the same response. No significantdifferences were observed in vessel sensitivity, pD2, either with dietgroup or type of animal. Red raspberry consumption for 8 wk restoredthe impaired vascular function of the OZR aorta associated withMetS.

Conclusion: Future studies should explore the vascular, molecularpathways that red raspberries target to confer their beneficial effects.

Funding SourcesFunding was provided by the National Processed Raspberry Coun-

cil. Red Raspberry powder was provided by FutureCeuticals (Momence,IL).

Vitamin D (VitD) Gummies as a Viable Alternative to Tablets:Results of Bioequivalence Comparative Studies in Healthy Adults(P08-118)

Carol L Wagner, Judy Shary, Amy H Wahlquist, Paul J Nietert,Myla D Ebeling, and Bruce Hollis

Medical University of South Carolina

Objective: The aim of this study was to compare the bioequivalenceof a single oral dose of vitamin D3 (vitD3) gummies and vitD3 tablets inhealthy adults.

Methods:Two separate institutional reviewboard–approved, single-center, crossover, randomized, comparator-controlled, single-blindclinical trials among healthy adults were conducted: Study 1 (pilot),n = 10; and Study 2, n = 31. Inclusion criteria were: body mass index18.5–29.9 kg/m2; 25(OH)D ≥25 ng/mL; nl serum calcium, and noevidence of anemia. At time 1, each subject received either 20,000 IUvitafusion Extra Strength VitD3 gummies (Church & Dwight Co., Inc.,Ewing, NJ) or Nature Made VitD3 tablets (Nature Made, Mission Hills,CA) with serial blood samples at baseline, 3, 6, 10, 24, and 48 h tomeasure vitD3 followed by a 2-wk washout period. At time 2, eachsubject received 20,000 IU vitD3 in the form not previously taken, withblood sampling at the same time points. The deidentified blood sampleswere processed, serum frozen, then sent for vitD3 analysis by LC-MS toHeartland Assays (Ames, IO). Data were analyzed with the use of SASversion 9.4 (Cary, NC).

Results: For Study 1, 9 subjects completed: 5 females and 4 males,mean± SD age 27.1± 3.7 y. Concentrationmax (Cmax) for both gummiesand tablets was achieved at 10 h (Figure 1). The geometric meanratios for AUC(time 0-t) and Cmax showed higher ratios with gummies(P = 0.003; P = 0.004). Log geometric mean ratios for AUCs(time 0-t)

remained significant (P = 0.003) as did log geometric mean ratios forCmax (P= 0.005). Similar results were found in Study 2 (Figure 2), which31 subjects completed: 14 males and 17 females, mean ± SD age of28.3 ± 5.6 y. Cmax at 10 h was 46.0 ng/mL (90% CI: 42.7, 49.6 ng/mL)in gummy compared with 22.9 ng/mL (90% CI: 21.4, 24.4 ng/mL) inthe tablet grp (P < 0.001). Geometric mean ratios for AUC(time 0-t) andCmax were higher with gummies (P= 0.001). Log geometricmean ratiosfor AUCs(time 0-t) remained significant (P < 0.001) as did log geometricmean ratios for Cmax (P< 0.001). Beyond baseline, gummymean valueswere significantly higher than those with tablets (P < 0.0001).

Conclusions: VitD3 gummies led to greater bioequivalence thandid tablets through better intestinal absorption as evidenced by greaterCmax at 10 h, higher circulating vitD3 concentration, and greater changefrom baseline at all tim epoints. When taking vitD supplements,enhanced intestinal absorption may have implications for achievingvitD sufficiency.

Funding SourcesResearch grant awarded to theMedical University of South Carolina

from Church & Dwight Co., Inc., Ewing, NJ.



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FIGURE P08-118-1 Time course of vitamin D3 concentrations in pilot study.

FIGURE P08-118-2 Time course of vitamin D3 concentrations in Study 2.

Dietary Fiber and Phenolic Compounds from Mango “Ataulfo”Pulp Exert Different yet Complementary Metabolic Effects in Ratswith Nonalcoholic Steatohepatitis (P08-119)

Abraham Wall-Medrano,1 J Abraham Domínguez-Ávila,2Jacqueline Ruiz-Canizales,2 Mónica A Villegas-Ochoa,2 NormaJ Salazar-López,3 and Gustavo González-Aguilar2

1Universidad Autonoma de Ciudad Juarez, Instituto deCiencias Biomedicas, Mexico; 2Centro de Investigación enAlimentación y Desarrollo AC, Mexico; and 3Universidad de Sonora,Mexico

Objective: The aim of this study was to evaluate thedifferential physiologic effects of dietary fiber (MF) and phenolic


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compounds (MP) isolated from freeze-dried mango cultivar“Ataulfo” pulp (FDM) in a nonalcoholic steatohepatitis (NASH) ratmodel.

Methods: Male albino rats (166 g) were fed for 12 wk with 5isoenergetic (3.4 kcal/g) diets (6 rats/diet): control, high cholesterol(0.8%)/sodium cholate (0.2%) alone (HCC), or supplemented with MF(6%), MP (0.1%), or FDM (5%). Serum lipids and cytokine levels,hepatic histomorphometry, fatty acid/cholesterol deposition and geneexpression (PPARα, LXRα, APOA1, and APOB mRNA levels) wereevaluated.

Results: The MP + HCC group showed a transiently higherAPOA1/APOB ratio in serum, but the hepatic gene expression ofboth proteins was similar among groups; although MF caused a lowerserum concentration of interleukin (IL)-1α/β , interferon (IFN)-γ , andtumor necrosis factor (TNF)-α, FDM increased IL-4, IL-6, and IL-10 concentrations. NASH was more pronounced in the HCC (alone)group, but all mango-supplemented diets reduced NASH to a differentextent (MP > MF > FDM), a fact associated with a lower cholesteroldeposition (–33%) and a different fatty acid profile. Ahigher PPARα andLXRα mRNA concentration was found in the MP- and MF-fed groups,respectively.

Conclusions: MP and MF exert synergistic hepatoprotectiveeffects in HCC-fed rats; such effects are more evident whenMP and MF were supplemented alone than combined(FDM).

Funding SourcesMexican Council of Science and Technology (CONACyT),

projects 563 (Un enfoque multidisciplinario de la farmacocinéticade polifenoles demango Ataulfo: Interacciones, moleculares, estudiospreclínicos y clínicos) and CB-2015-1/254063 (Antioxidantesde frutas y verduras en cáncer, ¿Son su bioaccesibilidad,biodisponibilidad o sinergismo estructural responsables de suineficacia?).

Effect of Eurotium cristatum Fermented Dark Tea Extract onBodyWeight and Blood Lipid in Rats (P08-120)

Liming Wang and Ting Liu

COFCO Nutrition and Health Research Institute

Objectives: Microbial fermentation mainly characterized by Eu-rotium cristatum is involved in the processing of dark tea, a traditionalChinese tea widely used since the Ming Dynasty (1500 AD), whichhas received increasing attention in recent years owing to its benefits,especially its antiobesity and hypolipidemic effects, to human health.

Methods: For this high-fat-diet–induced rat model 50 male SD ratswere randomly divided into 5 groups and consumed either normal or60% high-fat diet, and were given distilled water or the E. cristatumfermented dark tea extract by intragastric administration for 9 wk.Blood lipid was tested at baseline (week 1) and endpoint (week 9), andbody fat ratio was calculated from perirenal and epididymal fat weightat endpoint. Body weight, food intake, and energy intake were recordedweekly. Differentially expressed genes of rat liver were analyzed bygene microarray and real-time reverse transcriptase-polymerase chainreaction (RT-PCR).

Results: Body weight, serum triglycerides, food intake, and energyintake of the 250 and 750 mg/kg body wt E. cristatum fermented darktea extract groups were significantly decreased for the high-fat-diet–induced rat model. Gene microarray and real-time RT-PCR revealeda changed PPAR signaling pathway that might be a potent mechanismfor decreased body weight and serum triglycides.

Conclusion: The E. cristatum fermented dark tea extract decreasesbody weight and serum triglycerides for SD rats.

Funding SourcesThis research is financially supported by the Beijing Municipal

Science and Technology Project (grant Z161100000616012), China KeyR & D Project (2016YFD0400602) and the China National NativeProduce & Animal By-products Imp. & Exp. Corp., a wholly-ownedsubsidiary of COFCO.



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FIGURE P08-120-1

The Effect and Potential Mechanism of Lutein on Serum UricAcid in High-Fat-Fed Rats (P08-121)

Ling Wang,1 Haixia Li,1 Yongfang Zhao,2 and Ying Liu3

1Zhengzhou University, China; 2Renmin Hospital of HenanProvince, China; and 3Chinese Medical University of Henan Province,China

Objectives: The aims of this study were to investigate the effect oflutein on serum uric acid (SUA) in SD rats fed with high-fat diet andto explore the possible mechanisms of lutein on uric acid metabolismby comparing the enzyme levels of xanthine oxidase (XOD), adenosinedeaminase (ADA), superoxide dismutase (SOD), and the level ofmalondialdehyde both in serum and liver.

Methods: Fifty male SD rats aged 4 wk were randomly divided intoa control group and a high-fat diet group that consumed lab chow dietand high-fat diet, respectively, for 4 wk. Then the rats in the high-fat diet group were further divided into 4 groups (HF, HF + lutein L,

HF + lutein M, HF + lutein H). The rats in the lutein groups weregiven doses of 12.5, 25.0, and 50.0 mg · kg–1 · d–1 lutein by gavagefor 4 wk. The levels of SUA, TC, TG, high-density lipoprotein (HDL)cholesterol , serum creatinine (SCr), blood urea nitrogen (BUN), andmalondialdehyde (MDA) were measured. The activities of XOD, ADA,and superoxide dismutase (SOD), and MDA levels both in serum andliver were also measured.

Results: Before lutein gavage, the levels of SUA, TC, TG, and theactivity of XOD were significantly higher, whereas the serum HDLcholesterol was significantly lower in the HF group rats than in thecontrol group rats (P > 0.05). After 4 wk of gavage, the levels of SUA,TC, and MDA in the HF + lutein M and HF + lutein H groups weresignificantly lower, whereas the SOD activity was significantly higherthan that of the HF group (P

Conclusion: High-fat diet could induce hyperuricemia in rats.Lutein was hypouricemic at appropriate doses in hyperuricemic ratsthat consumed a high-fat diet. The hypouricemic effect of lutein


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might be due to improving lipid metabolism and reducing hepaticXOD activity, increasing antioxidant capacity, and reducing lipidperoxidation in rats fed with this high-fat diet.

Funding SourcesHenan Province Science and Technology Key Project.

Elenolic Acid Stimulates GLP-1 Secretion fromMouse Enteroen-docrine Cells through a Phospholipase C–Dependent Mechanism(P08-122)

Yao Wang and Dongmin Liu

Virginia Tech

Objective: Insulin resistance and β-cell dysfunction are the hall-marks of type 2 diabetes (T2D). The incretin hormone glucagon-like peptide-1 (GLP-1), which is secreted from intestinal L-cells, playsa critical role in maintaining glycemic homeostasis via potentiatingglucose-stimulated insulin secretion (GSIS) and promoting β-cellproliferation and survival. Therefore, targeting L-cells for promotingthe secretory function of GLP-1 could be an effective strategy fortreating andpreventingT2D.The present studywas aimed at identifyingbioactive small molecules targeting intestinal GLP-1 secretion toprevent and treat T2D. Here, we report that olive-derived elenolic acidis a potent agonist for GLP-1 secretion.

Methods: GLUTag L-cells and primary mouse ileum crypts wereincubated with various concentrations of elenolic acid. GLP-1 releasedinto supernatants was measured by an ELISA kit. Intracellular Ca2+

concentration was determined with the use of Fluo-4AM.Results: Elenolic acid dose-dependently stimulated GLP-1 release

in both clonal L-cells and primary ileum crypts, with 10 µM inducingmaximal effect. In addition, elenolic acid induced a rapid intracellularCa2+ increase. Inhibition of phospholipase C (PLC) ablated elenolicacid–stimulated intracellular Ca2+ release and GLP-1 secretion inL-cells. Consistently, elenolic acid elevated production of inositoltrisphosphate in the cells, indicating that elenolic acid activates PLC.Moreover, elenolic acid–triggered GLP-1 secretion from L-cells wasblocked by YM-254,890, a Gαq inhibitor. In vivo, oral administrationof elenolic acid tomice significantly reduced hyperglycemia and glucosetolerance in type 2 diabetic mice.

Conclusions: These data provide evidence that elenolic acid mightbe a novel antidiabetic agent through promoting endogenous GLP-1secretion.

Cancer Regulatory Proteins LKB1 and CaMKK2 Are Anti-thetically Regulated via the Activation of PKCzeta and DAPK,Respectively, by a Well-Defined Polyherbal Blend (PHB) (P08-123)

Jay Whelan,1 Amber MacDonald,1 Ahmed Bettaieb,1 DallasDonohoe,1 Anna Han,1 and Yi Zhao2

1University of Tennessee; and 2University of Michigan

Background: Liver kinase B1 (LKB1) is a tumor suppressor protein,and calcium calmodulin kinase kinase-2 (CaMKK2) is protumorigenicand overexpressed in many cancers. Activation of LKB1 is mediatedby the activity of protein kinase C-zeta (PKCzeta), whereas inhibitionof CaMKK2 occurs following phosphorylation at Ser511 by death-associated protein kinase (DAPK). DAPK activation appears to be an

important and universal inhibitor of the proliferation and promotionof a variety of cancers. Simultaneously, but antithetically, regulatingthese signaling proteins (LKB1 and CaMKK2) would be a tremendousadvancement in the treatment of cancer.

Objective: The aim of this study was to determine if a well-definedblend of 10 herbal extracts that has been successful in a variety of clinicaltrials could simultaneously regulate the activity of LKB1 and CaMKK2.

Methods: Upstream mediators (i.e., PKCzeta and DAPK), down-stream targets (i.e., AMPK), and both kinases (i.e., LKB1 andCaMKK2)were modulated through the use of pharmacologic and moleculartechniques in several cancer cell lines.

Results: The PHB increased the phosphorylation of PKCzeta,resulting in its translocation to the nucleus where it phosphorylated(Ser428) and activated LKB1. pLKB1 translocated to the cytosol whereit activated the tumor suppressor protein AMPK. Activation of AMPKresulted in suppression of cell proliferation. CaMKK2 was simulta-neously inhibited in the presence of the PHB via phosphorylation atSer511 mediated by DAPK. These results were verified in the presenceof chemical inhibitors (radicicol, STO-609, EGTA, BAPTA-AM, DAPKinhibitor, PKCzeta pseudosubstrate inhibitor), knockdown experimentsthat used siRNA techniques, and the use of an LKB1-null cell line thatoverexpresses CaMKK2, with or without the insertion of a wild-typeLKB1 construct or catalytically dead mutants of LKB1.

Conclusion: This PHB simultaneously, and antithetically, regulated2 parallel signaling pathways known to be important in cancer. This isthe first paper in the literature to report this kind of concurrent actioneither pharmacologically or with natural food products, and suggeststhat some natural products may have an advantage in cancer treatmentbecause of their simultaneous multiple targets of action.

Funding SourcesThis work was supported, in part, by a HATCH grant (TEN00441)

through the Tennessee Agricultural Experiment Station (JW), Univer-sity of Tennessee, Knoxville, TN, and by a grant from the NationalInstitutes for Health (R00DK100736) (AB).

Mass-BasedMetabolomic Analysis Reveals the Effects of a PrenylIsoflavonoid–Rich Extract from Erythrina variegata Linn on anOvariectomized Rat Model (P08-124)

Man-SauWong,1 Hui-Hui Xiao,2 Chi-On Chan,1 Si-Si Cao,1 andDaniel Kam-Wah Mok1

1The Hong Kong Polytechnic University; and 1State Key Laboratoryof Chinese Medicine and Molecular Pharmacology (Incubation), TheHong Kong Polytechnic University, Shenzhen Research Institute

Objectives: Erythrina variegata Linn (EV) is commonly used fortreatment of wind-damp syndrome in China, India, and SoutheastAsia. The present study aims to explore the alteration of endogenousmetabolites and identify the unique potential biomarkers that accountfor the bone-protective effects of a prenyl isoflavonoid–rich extract,containing 38 prenyl isoflavonoids, from EV.

Methods: Forty-eight female SD rats (3 mo old) were either sham-operated or ovariectomized (OVX). Upon recovery for 2 wk, the ratswere randomly assigned to the following groups (n = 12 each) andorally administered: vehicle-treated (Sham), vehicle-treated (OVX),Premarin-treated (PR, 130 mg/kg) and EV-treated (EV, 600 mg/kg)


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for 12 wk. Bone properties of distal femur, proximal tibia, and lumbarvertabra at L4 were measured with a Micro vivaCT 40 system; bonemetabolism–related genes were determined with real-time polymerasechain reaction; serum metabolomic profiles were analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flightmass spectrometry and the unique metabolic markers were identifiedby multivariate statistical analysis.

Results: Daily oral administration of the prenyl isoflavonoid–rich extract suppressed the OVX-induced increase in serum alkalinephosphatase (ALP) level and urine Ca excretion. Prenyl isoflavonoid–rich extract significantly increased bone mass and protected againstOVX-induced deterioration of bone microstructure in rats. Mostimportantly, unlike Premarin, EV extract did not increase uterusweight in OVX rats. In addition, EV extract significantly increasedmRNA expressions of the genes involved in bone formation (ALP andRunx2) and decreased mRNA expression of the gene involved in boneresorption (Ctsk) in tibia of OVX rats. Metabolomic analysis of ratserum indicated that 40 endogenous metabolites altered by EV extractwere restored to the normal level of Sham. Most of the identifiedmetabolites were related to steroid hormone metabolism and lipidmetabolism. Among them, 6 metabolites in OVX rats treated with EVextract were significantly higher than those in OVX rats treated withPremarin.

Conclusion: Our results indicated that the bone-protective effectsof the prenyl isoflavonoid–rich fraction of EV were associated with thechanges in steroid hormone metabolites and lipid metabolites in OVXrats.

Funding SourcesShenzhen Basic Research Program (JCYJ20151030164008764,

JCYJ20140819153305696)National Natural Science Foundation ofChina (NSFC, 81528024)

The Combination of Curcumin and Salsalate Suppresses High-Fat-Diet-Induced Procarcinogenic Signaling and Colorectal Tu-morigenesis in Azoxymethane-Treated Mice (P08-125)

Xian Wu, Gar Yee Koh, Jimmy Crott,and Joel Mason

Jean Mayer USDA Human Nutrition Research Center on Aging atTufts University, MA

Objective: High-fat diets (HFDs) and excessive adiposity elevateproinflammatory cytokines and activate signaling pathways that pro-mote colorectal cancer. Previously, we have shown that the combinationregimen of curcumin (CUR) and salsalate (SAL) suppresses biochem-ical inflammation and Akt/nuclear transcription factor κB (NF-κB)signaling in the colons of HFD-fed mice. Herein, we investigated thesuppression of signaling in greater detail, and determined whether thiscombination prevents the development of neoplastic tumors in thecolon.

Methods: Male A/J mice (n = 110) consumed a low-fat diet (LFD,10% kcal), a HFD (60% kcal), a HFD with CUR (0.4 wt%), SAL(0.3 wt%), or a combination of the two agents (CUR/SAL). All micereceived 6 injections of azoxymethane (AOM, 10 mg/kg), a colon-specific carcinogen.

Results: The HFD mice developed 31% greater fat mass than theLFD mice (P < 0.05). Colonic concentrations of interleukin (IL)-6 and

IL-1β were attenuated by the CUR/SAL combination (P < 0.02), butnot to a significant degree by either agent alone. CUR/SAL significantlysuppressed activation of PI3K/Akt/mTOR (P < 0.03) and NF-κB(P < 0.03) pathways, and upregulated AMPKα signaling (P < 0.02) inthe colonic mucosa. The adiposity generated by the HFD significantlyincreased tumor multiplicity and tumor burden in the HFD mice, andCUR/SAL also significantly reduced the development of these tumormetrics. Importantly, the combination was more effective than CUR orSAL alone in regard to suppressing tumor multiplicity and Akt/mTORsignaling, and activating AMPKα signaling.

Conclusion: These observations provide a scientific basis for futureclinical trials in obese subjects or those on HFDs.

Funding SourcesAgricultural Research Service and Tufts University Collaborates

grant.

Lycium barbarum Polysaccharides Ameliorate Blood Glucose,Protect Pancreas and Improve Liver Metabolism in High-Fat-Diet-and Streptozotocin-Induced Diabetic Rats (P08-126)

Hui Xia,1 Huali Tang,2 Feng Wang,2 and Guiju Sun2

1Key Laboratory of Environmental Medicine and Engineering ofMinistry of Education, and Department of Nutrition and Food hygiene,School of Public Health, Southeast University, China; and 2SoutheastUniversity, China

Objective:This work studies themetabolomics influences of Lyciumbarbarum polysaccharides (LBPs) on diabetic rats.

Methods: Twenty-four diabetic rats were randomly (6 rats/group)allocated to the diabetic control group (DC), the LBP low-dose group(50 mg · kg–1 · d–1) (LBP-L), the LBP middle-dose group (100 mg · kg–1· d–1) (LBP-M), and the LBP high-dose group (200 mg · kg–1 · d–1)(LBP-H). The normal rats were randomly allocated to a nondiabeticcontrol group (NDC) and a LBP high-dose group (NLBP-H) with 6 ratsin each group. After 30 d of LBP supplementation, blood, urine, andliver samples were collected for further biochemical detection, mainlyincluding oral glucose tolerance test, fasting blood glucose, insulin,liver glycogen, blood lipids, antioxidative stress variables, and livermetabolomics.

Results: LBP obviously moderated the fasting blood glucose andincreased the glucose tolerance of diabetic rat, compared with DC(P < 0.05). The weight of diabetic rats increased gradually within30 d among LBP supplementation groups, and at the end of theexperiment, only LBP-H showed a significant increase over DC with8.05 ± 3.97% versus –9.35 ± 3.34%. Furthermore, HbA1c levelsin plasma, homeostasis model assessment of insulin resistance, andinterleukin (IL)-8 levels decreased, and superoxide dismutase and liverglycogen levels increased significantly when compared with DC. Otherindexes were not observed to have significant changes. The differentmetabolites from the liver tissue between LBP-H and DC were l-malic acid, fumaric acid, d-arabitol, myo-inositol, l-allothreonine 1,xylitol, O-phosphorylethanolamine, ribitol, 5-methoxytryptamine 2,and digitoxose 2. Among these, only myo-inositol increased after LBPintervention; the other metabolites decreased.

Conclusion: This is the first study to apply metabolomics to observethe effects of LBP on diabetic rats. LBP can be an adjuvant therapy


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to help attenuate diabetic symptoms, improve insulin resistance, andincrease antioxidative stress and anti-inflammatory abilities throughoptimizing the distribution and change of metabolites in vivo.

Funding SourcesNational Key Research and Development Program of China

(2016YFD0400604-02), the National Natural Science Foundation ofChina (81273069), and the Fundamental Research Funds for theCentralUniversities (2242016K40024).

Development of AMPK Activators from Natural Products (P08-127)

Hong Yang,1 Hong Yang,1 Bin Lou,2 Lingling Zhao,1 DouglasStevenson,3 and Mark Bartlett3

1Nu Skin (China) Daily-Use & Health Products Co., Ltd, Centerfor Anti-Aging Research; 2Fudan University, China; and 3Nu SkinEnterprises

Objective: The aim of this study was to develop a purified enzyme-and cell-based assay to screen 5′AMP-activated protein kinase (AMPK)activators that upregulate AMPK activity from natural products.

Methods: The natural productsMomordica grosvenori (luo han guo,LHG) extract, sweet tea extract and epigallocatechin gallate (EGCG)were selected as model samples to establish the screening methodof the AMPK activity assay. Natural product sample–treated purifiedAMPK enzyme or HepG2 cell lysates were added to the well of aCycLex AMPK kinase assay kit (CycLex Co., Ltd, Japan) to test AMPKactivity according to the user’s manual, and then Western blotting withantibodies against phospho-ACC (acetyl-CoA carboxylase).

Results: The natural products LHG extract, sweet tea extract, andEGCG demonstrated an activation effect on purified AMPK enzyme

FIGURE P08-127-1 The effect of natural products on purified AMPK enzyme: luo han guo (LHG) extract (A), sweet tea extract (B), andepigallocatechin gallate (C) activated purified AMPK enzyme.

(Figure 1). Since the commercial purified AMPK enzyme is not stableand readily becomes inactive, we tried to set up the AMPK activity assayin a cell system. HepG2 cells were treated with or without LHG extract,sweet tea extract, and EGCG (C); these natural products were verified toactivate AMPK in the cultured cells. AICARwas referred to as a positivecontrol compound (Figure 2). Thus, the AMPK activity assay was set upbased on the HepG2 cell line.

Snow lotus tissue culturematerial and pepper extract were identifiedas AMPK activators in this established AMPK activity assay model,and the AMPK activation effects observed in the pepper and snowlotus samples were decreased in the presence of an AMPK inhibitor,compound C, in cultured cells (Figure 3). Then, the pepper and snowlotus samples were investigated on acetyl-CoA carboxylase activity(ACC) in liver cells. ACC is a downstream substrate of AMPK, therate-limiting enzyme of fatty acid synthesis. The phosphorylation ofACC reduced the fatty acid synthesis pathway. The pepper and snowlotus samples show an inhibitory effect on ACC activity by stimulatingphosphorylation of ACC (Figure 4).

Conclusion: We established a quick method to screen AMPKactivators from natural products, and identified pepper extract andsnow lotus tissue culture material as AMPK activators. They directlyactivate the enzyme AMPK in vitro, stimulate AMPK, and inhibit thekey enzyme ACC of fatty acid synthesis in the cell system. This makes itplausible to suppose that these two natural products exert a preventiveeffect on AMPK relevant metabolism regulation.

Funding SourcesResearch funding from theCenter for Anti-Aging Research, Nu Skin

Enterprises, Shanghai, China.



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FIGURE P08-127-2 AMPK activation by natural products in the cultured cells: : luo han guo (LHG) extract (A), sweet tea extract (B) andepigallocatechin gallate (C) activated AMPK in the cultured cells. AICAR was referred to as positive control compound.

FIGURE P08-127-3 Snow lotus tissue culture material (D) and Pepper extract (E) stimulated AMPK activity. The AMPK activation effectdue to snow lotus and pepper samples was decreased in the present of the AMPK inhibitor, compound C, in cultured cells.

FIGURE P08-127-4 Snow lotus tissue culture material (D) and pepper extract (E) inhibited acetyl-CoA carboxylase (ACC) activity bystimulating phosphorylation of ACC in liver cells.


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Piceatannol Attenuates Fat Accumulation in Steatosis-InducedHepG2 Cells (P08-128)

Jason S Yang,1 Jozxelle Tongson,1 Kee-Hon Kim,2 and YeonhwaPark1

1University of Massachusetts, Amherst; and 2Purdue University, IN

Objective: Nonalcoholic fatty liver disease (NAFLD) is the mostcommon liver disease worldwide, and affects >20% of the adultpopulation; it can progress to inflammatory hepatitis, cirrhosis, andliver cancer. However, due to limited pharmacologic therapies, theneed to alleviate NAFLD is imperative. Based on previous reports thatpiceatannol, a stilbenoid metabolite of resveratrol found in grapes andpassion fruit, exhibits antiobesity and anti-inflammatory effects, thegoal of this study was to determine the efficacy of piceatannol on theprevention and treatment of NAFLD.

Methods: HepG2 human hepatocytes were used as an in vitroNAFLD model. Steatosis was induced by 600 mM fatty acid (FAs)mixture (oleic acid:palmitic acid, 2:1). Cell viability was determined byMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)tetrazolium reduction assay. Fat accumulation was determined bytriglyceride assay. Protein and mRNA expressions were evaluated byconducting Western blot and real-time quantitative polymerase chainreaction, respectively.

Results: Piceatannol was not cytotoxic for up to 100 mM for 24h with or without FA treatments. Compared with the FA-only group,100 mM of piceatannol significantly decreased fat accumulation inHepG2 cells (19.6%, P = 0.0002). Under steatosis conditions, piceatan-nol suppressed genes for lipogenesis and FA uptake, notably sterolregulatory element–binding protein 1 (SREBP1, 53%, P = 0.0111) andcluster of differentiation 36 (CD36, 53%, P = 0.0039) compared withthe FA-only group. It also promoted genes for FA oxidation, peroxisomeproliferator–activated receptor α (PPARα, 62%, P < 0.0001) andcarnitine palmitoyltransferase Iα (CPT1α, 170%,P< 0.0001) comparedwith the FA-only group.

Conclusions: Piceatannol may reduce fat accumulation in steatosis-induced HepG2 cells by suppressing lipogenesis (SREBP1) and FAuptake (CD36), and promoting FA oxidation (PPARα and CPT1α).

Simultaneous Determination of Eight Nonvolatile Ginger Con-stituents in Dietary Supplements and Ingredients by HPLC withdiode array detection (P08-129)

Hong You,1 Bailey Ireland,1 Michael Moeszinger,1 Laura Snow,2Scott Krepich,2 Vivian Takagawa,3 and Darlene Enriquez1

1Eurofins Scientific, Inc.; 2Phenomenex; and 3ChromaDex

Objective: Nonalcoholic fatty liver disease (NAFLD) is the mostcommon liver disease worldwide, and affects >20% of the adultpopulation; it can progress to inflammatory hepatitis, cirrhosis, andliver cancer. However, due to limited pharmacologic therapies, theneed to alleviate NAFLD is imperative. Based on previous reports thatpiceatannol, a stilbenoid metabolite of resveratrol found in grapes andpassion fruit, exhibits antiobesity and anti-inflammatory effects, thegoal of this study was to determine the efficacy of piceatannol on theprevention and treatment of NAFLD.

Methods: HepG2 human hepatocytes were used as an in vitroNAFLD model. Steatosis was induced by 600 mM fatty acid (FAs)mixture (oleic acid:palmitic acid, 2:1). Cell viability was determined byMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)tetrazolium reduction assay. Fat accumulation was determined bytriglyceride assay. Protein and mRNA expressions were evaluated byconducting Western blot and real-time quantitative polymerase chainreaction, respectively.

Results: Piceatannol was not cytotoxic for up to 100 mM for 24 hwith or without FA treatments. Compared with the FA-only group, 100mM of piceatannol significantly decreased fat accumulation in HepG2cells (19.6%, P = 0.0002). Under steatosis conditions, piceatannol sup-pressed genes for lipogenesis and FA uptake, notably sterol regulatoryelement–binding protein 1 (SREBP1, 53%, P = 0.0111) and clusterof differentiation 36 (CD36, 53%, P = 0.0039) compared with theFA-only group. It also promoted genes for FA oxidation, peroxisomeproliferator–activated receptor α (PPARα, 62%, P < 0.0001) andcarnitine palmitoyltransferase Iα (CPT1α, 170%,P< 0.0001) comparedwith the FA-only group.

Conclusions: Piceatannolmay reduce fat accumulation in steato-sisinduced HepG2 cells by suppressing lipogenesis (SREBP1) and FAuptake (CD36), and promoting FA oxidation (PPARα and CPT1α).

Funding SourcesCompanies’ internal research and development funding.

Blueberry Leaf Extract Inhibits Foam Cell Formation and Acti-vates Autophagy in Macrophages via AMPK/mTOR Signaling (P08-130)

MD Khurshidul Zahid,1 Masao Yamasaki,2 Teruaki Arakawa,3and Shaikh Mizanoor Rahman1

1Texas Tech University; 2University of Miyazaki, Miyazaki, Japan;and 3Bizen Chemical Co. Ltd, Okayama, Japan

Objectives: Atherosclerosis is characterized by macrophage foamcell formation and abnormal lipid metabolism and is the leading causeof death in Western societies. Evidence now suggests that the foamingof macrophages is a crucial early step in the development of atheroscle-rosis. Autophagy is a catabolic process that removes unfolded proteinsand damaged organelles and helps to main cellular homeostasis. Recentstudies have shown that autophagy activation increases cholesterolefflux, inhibits inflammation, and prevents atherosclerosis. Blueberryis a polyphenol-rich fruit with known health benefits. However, thephysiologic functions of bioactive compounds present in blueberry leaf(BBL) remain unexplored. The present study explored the effects ofBBL extract on foam cell formation, autophagy activity, and cholesterolhomeostasis in lipid-loaded macrophages.

Methods: RAW 264.7 and bone marrow–derived macrophages(BMDMs) were treated without (control) and with lipopolysaccharide(LPS; 100 ng/mL) and oxidized low-density lipoprotein (oxLDL;25 µg/mL) for 6–24 h followed by treatment with BBL (10 and 25µg/mL) for an additional 6–24 h. Expression of proteins and geneswere analyzed by Western blot and quantitative reverse transcriptase-polymerase chain reaction, respectively. Oil red O staining was em-ployed to detect lipid accumulation. Statistical analysis was performedby either Student’s t test or ANOVA.


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Results: Treatment with BBL extract attenuated LPS- and oxLDL-mediated induction of foam cell formation in both RAW264.7 cellsand BMDMs. Additionally, BBL extract significantly reduced theexpression of activating transcription factor 4 (ATF4) and C/EBPhomologous protein (CHOP) in macrophage foam cells. Interestingly,expression of proteins implicated in cholesterol efflux (ABCA1) andautophagy (LC3II andATG5) were significantly induced in BBL-treatedmacrophages. More importantly, treatment with BBL extract increasedAMPK phosphorylation and reduced mTORC1 gene expression, twokey molecules responsible for autophagy regulation, in LPS- andoxLDL-loaded macrophages.

Conclusion: Our study indicates that BBL extract attenuates foamcell formation, inhibits apoptosis, and induces autophagy activity bytargeting AMPK and mTOR in LPS- and oxLDL-loaded macrophages.Thus, BBL extract may be an important therapeutic option to preventand treat atherosclerosis.

Funding SourcesStartup Fund, Texas Tech University.

Soyasaponins Reduce Inflammation and Improve Lipid andGlucose Homeostasis in High-Fat-Diet–Induced Obese Mice(P08-131)

Longying Zha, Qunying Xie, Xiangfu Gu, Junbin Chen, , MinshuLiu, Fei Xiong, Xinglong Wu, Yajie Zhang, Fengping Chen,Honger Chen, Meijua Li, Suxia Sun, and Xinwei Chu

Southern Medical University, China

Objectives: Obesity is causatively linked to a chronic low-gradeinflammatory state that contributes to the development of metabolicdisorders. The effects of soyasaponins (SS) on chronic inflammationand metabolic disorders in obesity are not fully understood.

Methods: Seventy-five high-fat-diet (HFD)–induced obese maleC57BL/6J mice were randomly allotted to 5 groups [HFD control,HFD + 0.1 mg/kg body weight (BW) of aspirin (ASP), HFD + 20mg/kg BW of SS-A1, SS-A2, or SS-I, respectively] and treated for 8weeks. Another age-matched 15 mice consuming chow diet were usedas negative control. Systemic and tissue inflammation, BW, and bloodindexes of lipids and glucose were monitored.

Results: SS andASP did not affect BWand feed intake in obesemice.ASP lowered tumor necrosis factor α (TNF-α), interleukin 6 (IL-6),monocyte chemoattractant protein-1 (MCP-1), nitrite oxide (NO), andprostaglandin 2 (PGE2) in serum. SS (A1,A2, I) decreasedTNF-α,MCP-1, and NO. Furthermore, SS-A1 decreased IL-6, leptin and PGE2, SS-A2

reduced leptin, and SS-I lowered PGE2. Both ASP and SS (A1, A2, I)increased adiponectin. ASP lowered total cholesterol (TC), triglyceride(TG), and elevated high-density lipoprotein (HDL) cholesterol, but didnot affect low-density lipoprotein (LDL) cholesterol in serum. SS (A1,A2) decreased TC, TG, and LDL cholesterol. SS-I reduced TG and LDLcholesterol. All SS (A1, A2, I) increased HDL cholesterol. The fecalexcretions of cholesterol and bile acids were enormously increased by SS(A1, A2, I), but not by ASP. ASP and SS-A2 improved glucose toleranceand decreased fasting blood glucose, insulin, and homeostasis modelassessment of insulin resistance (HOMA-IR). SS-A2 enhanced HFD-blunted Akt phosphorylation in muscle and liver. Interestingly, SS (A1,A2, I) all enhanced Akt phosphorylation in white adipose tissue (WAT).

All SS (A1, A2, I) alleviated HFD-induced steatosis and inflammationin liver. ASP and SS (A1, A2, I) inhibited IKKа/β phosphorylationin liver. ASP and SS (A1, A2) also suppressed nuclear transcriptionfactor κB (NF-κB) p65 phosphorylation in liver. SS (A1, A2) reducedthe adipocyte hypertrophy in WAT. ASP and all SS (A1, A2, I) de-creased IKKа/β and NF-κB p65 phosphorylation and CD68 expressionin WAT.

Conclusions: SS can reduce inflammation and improve lipid andglucose homeostasis in HFD-induced obese mice.

Funding SourcesThis work was supported by grants from National Natural Science

Foundation of China (NSFC) (81573125 andNo.81102130), theNaturalScience Foundation ofGuangdong Province, China (2014A030313313),and the Research Start-up Plan of Southern Medical University(CX2016N012).


FIGURE P08-131-1 Soyasaponins improve glucose dysfunction.

FIGURE P08-131-2 Soyasaponins improve lipid profiles in serum.


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FIGURE P08-131-3 Soyasaponins reduce adipocyte hypertrophy in white adipose tissue.

FIGURE P08-131-4 Soysaponins decrease liver steatosis and inflammation.

Ubiquinol is a Better Form than Ubiquinone for EnhancingCoenzyme Q10 Status in Older Men (P08-132)

Ying Zhang,1 Xiao-qiang Chen,1 Jin Liu,2 and C-Y Oliver Chen2

1Northeast Forestry University, China; and 2Tufts University, MA

Objective: Coenzyme Q10 (CoQ10) exerts its functions in thebody through the ability of its benzoquinone head group to acceptand donate electrons. The primary functions are to relay electronsfor the ATP production in the electron transport chain and to act asan important lipophilic antioxidant. Ubiquinone, the oxidized formof CoQ10, is commonly formulated in commercial supplements, butit must be reduced to ubiquinol to exert CoQ10’s functions afterconsumption. Thus, we aimed to examine whether ubiquinol would

be more effective than ubiquinone at enhancing CoQ10 status in oldermen.

Methods:Weconducted a double-blind, randomized, crossover trialwith two 2-wk intervention phases and a 2-wk washout between the2 phases. Ten eligible older men [>55 y, bodymass index: 25–35 kg/m2,and

Results: After 2 wk of the supplementation, the ubiquinol supple-ment significantly increased plasma ubiquinol status by 1.5-fold from1.1 to 2.8µmol/L, plasmaubiquinone by 1.7-fold from0.2 to 0.6µmol/L,and total CoQ10 (the sumof 2 forms) by 1.5-fold from 1.3 to 3.4µmol/L(P

Conclusions: The significant increase in plasma ubiquinol,ubiquinone, and total CoQ10 observed after the 2-wk supplementation


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suggest that ubiquinol appeared to be a better supplemental form toenhance the CoQ10 status than ubiquinone in older men. Neitherubiquinol nor ubiquinone supplement affects the measured biomarkersof oxidative stress.

Funding SourcesKaneka and USDA.

Flavonoids from Chinese Bayberry Leaves Inhibit Ovarian Can-cer Cells by Inducing Apoptosis and G1 Cell Cycle Arrest via ErkSignaling (P08-133)

Yu Zhang,1 Xingqian Ye,1 Yi Charlie Chen,2 and Shiguo Chen1

1Zhejiang University, China; and 2Alderson Broaddus University,WV

Ovarian cancer is one of the leading causes of death related tothe female reproductive system in Western countries. Patients givenplatinum-based chemotherapy might suffer some side effects andacquire resistance to treatment drugs, which is the major impedimentfor ovarian cancer treatment. In recent years, natural products haveattracted great attention in the fight against cancer, and flavonoids havebeen reported to exhibited anticancer functions in various signalingpathways. However, studies about the effects of flavonoids on cisplatin-resistant ovarian cancer cells are scarce. Chinese bayberry (Myrica rubraSieb. et Zucc.) has been cultivated in southern China for >2000 yand is popular among local people. However, leaves from bayberrytrees are always abandoned after harvest, which causes huge ecologicalwaste, and further utilization and development of this material hasbeen sought. Flavonoids from Chinese bayberry leaves (BLF) containmyricitrin and quercetin 3-rhamnoside as major components, andexhibit strong antioxidant properties based on the chemical and cellular

FIGURE P08-133-1 Cell viability

assays from a previous study from our group. In the present study,we further investigated the effects of BLF on a cisplatin-resistantovarian cancer cell line A2780/CP70. BLF showed strong inhibitoryeffects on the growth of A2780/CP70 cells, and such effects mightbe attributed to the BLF-induced apoptosis and G1 cell cycle arrest.BLF treatment increased the expression of cleaved caspase-3 and -7, and induced apoptosis via an Erk-dependent caspase-9 activationintrinsic apoptotic pathway by upregulating the proapoptotic proteins(Bad and Bax) and downregulating the antiapoptotic proteins (Bcl-xLand Bcl-2). Furthermore, by reducing the expression of cyclin D1 andCDK4 and p-Erk, BLF elevated the distribution of the G1 phase of thecell cycle. BLF exhibited its anticancer property by mainly targetingthe Erk pathway, which is quite different from other flavonoids thataffect various signaling pathways and might possibly cause more sideeffects. Overall, these results indicated that BLF has the potential to bedeveloped as a valuable anticancer agent to promote public health.

Funding SourcesThis research was supported by NIH grants P20RR016477 from the

National Center for Research Resources and P20GM103434 from theNational Institute for General Medical Sciences (NIGMS) awarded tothe West Virginia IDeA Network of Biomedical Research Excellence.This research was supported by grant P20GM104932 from NIGMS,a component of the National Institutes of Health (NIH) and itscontents are solely the responsibility of the authors and do notnecessarily represent the official view of NIGMS or NIH. This studywas also supported by COBRE grant GM102488/RR032138, ARIA S10grant RR020866, FORTESSA S10 grant OD016165 and INBRE grantGM103434. This research was also supported by the National NaturalScience Foundation of China (C200501) and the National Key Researchand Development Program (2016YFD0400805).



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FIGURE P08-133-2 Flow cytometry and staining.


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FIGURE P08-133-3 Caspases


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FIGURE P08-133-4 Intrinsic and extrinsic.


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FIGURE P08-133-5 Cell cycle.

Inhibitory Effects Of Green Tea Polyphenols on MicrobialMetabolism of Aromatic Amino Acids in Humans Revealed byMetabolomic Analysis (P08-134)

Yuyin Zhou,1 Ningning Zhang,1 Andrea Arikawa,2 and ChiChen,1

1University of Minnesota-Twin Cities; and 2University of NorthFlorida

The bioactivities of green tea polyphenols (GTPs) on cell pro-liferation and survival have been extensively investigated, but themetabolic impacts of chronic GTP intake on humans are not welldefined. In this study, fecal and urine samples from postmenopausalfemale subjects taking the GTP supplement or placebo for 12 mowere analyzed by LC-MS–based metabolomics analysis. The GTP-responsive metabolites were identified and characterized by structuralelucidation and quantitative analysis of the metabolites, contributing


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to the separation of control and treatment samples in the multivariatemodels.Major GTPs and their sulfate and glucuronidemetabolites werenot found in significant amounts in feces and urine. In contrast, GTP-derived phenolic acids were identified as the robust exposuremarkers ofGTPs, suggesting extensivemicrobial biotransformation ofGTPs. Inter-estingly, GTPs decreased the levels of microbial metabolites of aromaticamino acids, including indoxyl sulfate and phenylacetylglutamine inurine and indole-3-carboxaldehyde in feces, but did not affect the levelsof aromatic amino acids in feces.Othermicrobialmetabolites, includingshort-chain fatty acids and secondary bile acids, were not affectedby GTPs. Overall, these results suggest that microbial metabolism ofaromatic amino acids in human gut might be selectively inhibited bychronic GTP consumption.

Funding SourcesNCI grant R01 CA127236NIFA project MIN-18-092.

Effects of Dietary Avicularin on Gut Microbiota in Mice (P08-135)

Xiaoai Zhu,1,2 Min Gu,2 Mingyue Song,1,2 Yanhui Han,2 Xiao-qiong Cao,2 Yong Cao,1,2 and Hang Xiao2

1South China Agricultural University, China; 2University of Mas-sachusetts Amherst

Objectives: Gut microbiota play an important role in host health,and dysbiosis of gut microbiota is related to many diseases, such asobesity, inflammation, diabetes, and cancer. Accumulating evidenceindicates that the biological activities of dietary polyphenols are relatedto their effect on modulating the composition of gut microbiota. The

aim of this study was to determine the effects of dietary intake ofavicularin, a plant-derived polyphenol commonly found in apple andmango, on the composition of gut microbiota in CD-1 mice.

Methods: Male CD-1 mice consumed avicularin in diet (0.1%wt/wt), and their body weight, food and water intake, and bloodchemistry parameters were monitored for 3 wk. 16S rRNAgene sequencing was utilized for the analysis of gut microbiotacommunity.

Results: The monitoring indexes during the entire experimentalperiod demonstrated that dietary intake of avicularin did not causeany noticeable adverse effects in the mice. Results of 16S rRNA genesequencing showed that dietary treatment with avicularin modulatedthe composition of gut microbiota by significantly reducing theabundance of g_ucispirillum (P < 0.05), the prevalence of whichwas associated with increased inflammation, and decreasing theabundance of 2 genera of Firmicutes (P < 0.05) that were po-tentially related to obesity. Furthermore, the levels of short-chainfatty acids in colonic contents were greatly increased by dietaryavicularin.

Conclusions:Overall, these results indicated that the dietary intakeof avicularin may have beneficial effects on improving homeostasis ofgut microbiota.

Funding SourcesThe authors are grateful for the scholarship provided by the China

Scholarship Council. This work was financially supported by theUniversity of International Cooperation in Science and TechnologyInnovation Platform Project of Guangdong (grant 2013gjhz0003), theNational Natural Science Foundation of China (31201321), and theeducation department of Hunan Province (B13084).


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dietary bioactive components

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