ResearchArticle Vaccinium macrocarpon Aiton Extract...

Research ArticleVaccinium macrocarpon Aiton Extract AmelioratesInflammation and Hyperalgesia through Oxidative StressInhibition in Experimental Acute Pancreatitis

Danielle Gomes Santana1 Alan Santos Oliveira1 Mariacutelia Trindade de Santana Souza1

Joseacute Thiago do Carmo Santos23 Neuza Mariko Aymoto Hassimotto23

Ana Mara de Oliveira e Silva4 Renata Grespan 1 and Enilton Aparecido Camargo 1

1Department of Physiology Federal University of Sergipe 49100-000 Sao Cristovao SE Brazil2Department of Food Science and Experimental Nutrition School of Pharmaceutical Sciences University of Sao Paulo05508-000 Sao Paulo SP Brazil3Food Research Center (FoRC) Research Innovation and Dissemination Centers-Sao Paulo Research Foundation (CEPID-FAPESP)05508-080 Sao Paulo SP Brazil4Department of Nutrition Federal University of Sergipe 49100-000 Sao Cristovao SE Brazil

Correspondence should be addressed to Enilton Aparecido Camargo eniltoncamargopqcnpqbr

Received 28 February 2018 Accepted 4 April 2018 Published 14 May 2018

Academic Editor Letizia Angiolella

Copyright copy 2018 Danielle Gomes Santana et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

We evaluated the effect of the hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) in a model of acute pancreatitis(AP) in mice AP was induced by two injections of L-arginine and animals were treated with HEVm (50 100 and 200mgkgpo) or vehicle (saline) every 24 h starting 1 h after the induction of AP Phytochemical analysis of the extract and measurementof inflammatory and oxidative stress parameters as well as abdominal hyperalgesia were performed Catechin epicatechin rutinand anthocyanins were identified in HEVm Treatment with HEVm decreased L-arginine-induced abdominal hyperalgesia (from48 to 72 h) Also treatment with HEVm decreased L-arginine-induced pancreatic edema pancreatic and pulmonary neutrophilinfiltration and levels of tumor necrosis factor-120572 interleukin-1120573 and interleukin-6 after 72 h of induction L-arginine-inducedhyperamylasemia and hyperlipasemia were also reduced by the treatment with HEVm in comparison to vehicle-treated groupMoreover lipoperoxidation carbonyl radicals nonprotein sulfhydryl groups and activity of catalase and superoxide dismutase butnot glutathione peroxidase were restored by the treatment with HEVm These results show that treatment with HEVm decreasedhyperalgesia and pancreaticextrapancreatic inflammation and oxidative damage in L-arginine-induced AP making this extractattractive for future approaches designed to treat this condition

1 Introduction

Acute pancreatitis (AP) is a disease that leads to pancreaticinflammation related to the premature activation of digestiveenzymesThe incidence of this disease has increased in recentyears [1] which has contributed to a significant increase inmortality mainly caused by complications of this conditionsuch as the systemic inflammatory response [2]

The treatment of AP is primarily symptomatic and basedon the initial evaluation of disease severity It is directed to

control pain promote supportive therapy meet the nutri-tional needs treat complications and prevent pancreaticinfection and spread of the inflammatory process to remoteorgans [3] The lack of specific therapy for AP along withthe high mortality rates observed in severe cases makesexperimental studies to find new treatment alternatives ofgreat interest

Among the sources of new medicinal active principlesnatural products with proven antioxidant potential are ofinterest for the treatment of AP since the local release

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 9646937 13 pageshttpsdoiorg10115520189646937

2 Evidence-Based Complementary and Alternative Medicine

of reactive oxygen species (ROS) and inflammatory cellinfiltration in the pancreas play a key role in the developmentof AP and complications associated with this condition [4]

The fruit ofVacciniummacrocarpon Aiton (Ericaceae) ispopularly known as cranberry and is used to make juices andsauces throughout North America Due to promising resultsthe medicinal use of this plant has spread throughout tothe Western world [5] It contains anthocyanins flavonoidsproanthocyanidins condensed tannins and phenolic acidsRecent studies have shown that an extract of fruits of Vmacrocarpon containing approximately 182 anthocyaninscan attenuate hepatic inflammation in obese mice [6] andinhibit the adhesion of pathogenic E coli in patients withrecurrent urinary tract infection [7] In addition treatmentwith this extract led to decreased intestinal inflammation inobese mice [5] Clinically cranberry preparations have beenused in the treatment of urinary tract diseases and vascularinflammation demonstrating high therapeutic potential [8]which may still be underestimated

The need for new therapeutic options to treat AP alongwith the potential anti-inflammatory and antioxidant effectpresented by cranberry makes it a viable alternative for thetreatment pancreatitis Thus we investigated the possibleeffect of a hydroethanolic extract of fruits of Vacciniummacrocarpon (HEVm) in experimental AP in mice

2 Material and Methods

21 Plant Material The hydroethanolic extract of fruits ofVacciniummacrocarpon (HEVm)was purchased fromFagronBV lot number 14083757D with certificate of quality controlanalysis (HHHC140402)

22 Characterization of Hydroethanolic Extract of Fruitsof Vaccinium macrocarpon Samples were dissolved withmethanol water (70 30 v v) acidified with 5 acetic acidusing an ultrasound bath (Ultrasonic Cleaner 8892E-DTHCole-Parmer Illinois USA) After 10min at room temper-ature the dissolved extract was centrifuged at 3500timesg for10min at 4∘C This procedure was repeated 3 times and thesupernatants were combinedThe supernatants were reducedunder vacuum at 40∘C in a rotary evaporator (RotavaporRE 120 Buchi Flawil Switzerland) prior to passage througha C-18 extraction column (1 g) (Supelclean LC-18 SupelcoPennsylvania USA) previously conditioned with methanoland ultrapure water The sample was loaded into the columnand washed with ultrapure water The phenolic compoundswere eluted with methanol acidified with 03 HCl Theeluateswere completely dried using a rotary evaporator undervacuum at 40∘C resuspended in methanol acidified with5 acetic acid and filtered through a 045 120583m PVDF filter(Millipore Ltd) for the quantification of flavonoids by high-performance liquid chromatography-diode array detection(HPLC-DAD) analysis

Quantification of flavonoidswas carried out using anAgi-lent 1260 Infinity Quaternary LC System (Agilent Technolo-gies USA) coupled to a diode array detector (DAD)The col-umnusedwas a 5 120583mProdigyODS3 column (460times 250mm)(Phenomenex Ltd UK) with a flow rate of 1mLmin at 25∘C

The mobile phase consisted of waterformic acidacetonitrile(96 1 3 solvent A and 58 1 51 solvent B) The solventgradient was 10 B at the beginning 25 at 10 minutes31 at 15 minutes 40 at 20 minutes 50 at 30 minutes100 at 40 minutes 10 at 45 minutes and 10 at 50minutes Chromatograms were acquired at 270 370 and525 nm The flavonoids were identified by comparing theirretention times diode array spectral characteristics andmassspectra analyzed using LC-ESI-MSMS with the standardsand the data available in the literature The quantificationwas done using a calibration curve of the commercial stan-dards (catechin epicatechin from Sigma-Aldrich (St LouisMO USA) and cyanidin-3-O-glucoside from ExtrasyntheseGenay France)

The identification of the flavonoids was performed usinga Prominence Liquid Chromatograph (Shimadzu Japan)linked to an AmaZon SL ion trap mass spectrometer (BrukerDaltonics BremenGermany)with an electrospray ionization(ESI) interfaceThe solvent gradient conditionswere the sameas those used for the HPLC-DAD analysis After the samplewas passed through the DAD the flow rate was changedto 08mLmin for examination by the mass spectrometerThe ESI was used in the positive and negative mode Themass spectrometer operating condition was collision energyof 4500V for positive and negative mode The analysiswas carried out using a full scan from 119898119911 100 to 1500The compounds were identified according to mass spectralcharacteristics and comparison with literature data

23 Animals Male adult Swiss mice (30ndash35 g) were obtainedfrom the Animal Center of Federal University of SergipeThe animals were maintained at 21 plusmn 2∘C with free access tofood (Nuvilab) and filtered water under a 12 12 h lightdarkcycle All experimental procedures were approved by theEthics Committee for Animal Use in Research of FederalUniversity of Sergipe (protocol number 0916) and wereconducted in compliance with the Guide for Care and Use ofLaboratory Animals (National Institutes of Health) Duringthe experiments animals were randomly distributed amongthe experimental groups and the experimenter was unawareof each grouprsquos identification

24 Experimental Procedure Pancreatitis induction was per-formed by two intraperitoneal (ip) injections 1 h apartof L- arginine at the dose of 4 gkg [9] The solution forinjection contained 8 L-arginine in sterile saline with pHadjusted to 70 The control group received sterile saline(NaCl 09) Seventy-two hours after the last injectionthe animals were anesthetized (80mgkg of ketamine plus10mgkg of xylazine) euthanized by exsanguination andtranscardially perfused with saline plus heparin (5 IUL) andtissue samples (pancreas and lung) were collected Blood wascentrifuged at 1000timesg for 15min at room temperature toseparate serum samples Aliquots of serum and tissues werestored at minus80∘C for proper analysis

25 Experimental Design Animals were randomly dis-tributed into five groups (119899 = 5-6 each) In the saline +vehicle group animals were injected with sterile saline (09

Evidence-Based Complementary and Alternative Medicine 3

5mLkg ip administration for 2 times) and treated with thevehicle (sterile saline ip) at 1 25 and 49 hours after the lastinjection of saline In the L-arginine + vehicle group animalswere injected with L-arginine (4 gkg ip administration for2 times) and treated with the vehicle (ip) at 1 25 and 49hours after the last injection of L-arginine In the three AP +HEVmgroups animals were injectedwith L-arginine (4 gkgip administration for 2 times) and treated with HEVm (50100 or 200mgkg po) at 1 25 and 49 hours after the lastinjection of L-arginine

Additional control groups were treated with dexametha-sone (5mgkg sc) at 1 25 and 49 hours after the last injec-tion of L-arginine (control for inflammatory parameters) ormorphine (5mgkg ip 30min before each measurementperformed at 24 48 and 72 h control for abdominal hyper-algesia) [10]

26Measurement of AbdominalHyperalgesia To evaluate themechanical hyperalgesia in the abdominal region of micewe used an electronic von Frey apparatus (Insight RibeiraoPreto Sao Paulo Brazil) according to Abreu et al [11] withminor adaptations On the day of testing mechanical stimuliwere applied to the anterior lateral region of the abdomenof the animals in triplicate prior to any manipulation and24 48 or 72 h after AP induction At each time point anincreasing stimulus (in g) was applied to the abdominalregion of themice with an interval of at least 1min apart untilany withdrawal behavior was observed and the thresholdforce (01 to 1000 g) value was registered by the equipment

This test was performed off-stage so that the examinerdid not know the identity of the groups Data were expressedas variation (Δ) by subtracting the mean value obtained fromthe three measurements taken at the time point referred toafter AP induction from the mean basal value recorded foreach animal (prior to pancreatitis)

27 Evaluation of Locomotor Activity The locomotor activityof mice was investigated in a circular open field apparatus(60 cm diameter Insight Ribeirao Preto Sao Paulo Brazil)as previously described [12] The animals (119899 = 5) werepretreated with HEVm (200mgkg) or vehicle (1 h before)or diazepan (1mgkg 30min before) and were individuallyplaced in the apparatus After 1min the distance traveled (m)mobility time (s) and number of lines crossedwere registeredover a period of 5min

28 Determination of Inflammatory Markers The edemaindex was calculated as the ratio of wet weight and dry weightof the samples of pancreatic tissue as previously described[10]

To determine myeloperoxidase (MPO) activity pancreasand lung samples were collected and homogenized withpotassium phosphate buffer (50mM pH 60 containing05 hexadecyltrimethylammonium bromide) Aliquots ofthe homogenates were centrifuged (2min 8000timesg 4∘C)and aliquots of the supernatants were incubated with a solu-tion of o-dianisidine hydrochloride (0167mgmL containing0005H2O2)TheMPOactivity wasmeasured kinetically inamicroliter plate scanner (Biotek) at 460 nmover a period of

5min Results were expressed as units of MPO per mg tissue(UMPOmg tissue) UMPOwas considered as the amount ofenzyme that degrades 1mmol of hydrogen peroxidemin [13]

Enzyme-linked immunosorbent assay (ELISA) forinterleukin- (IL-) 6 IL-1120573 and tumor necrosis factor-(TNF-) 120572was carried out in homogenate samples of pancreasand lung according to the manufacturerrsquos instructions (RampDSystems) The results were corrected by the amount oftissue protein and expressed as pg of each cytokinemg ofproteinThe protein content of tissues was determined by theBradford method using the Bio-Rad protein assay reagent

29 Determination of Biochemical Parameters in SerumBiochemical parameters in serum were assessed for eachexperimental group by using specific commercial kits foramylase (Katal Biotechnology Belo Horizonte MG Brazil)lipase (Human do Brasil Sao Paulo SP Brazil) aspar-tate aminotransferase (AST) and alanine aminotransferase(ALT) (Liquiform Labtest Lagoa Santa MG Brazil) accord-ing to each manufacturerrsquos instructions

210 Determination of Antioxidant Activity Samples of pan-creas and lungwere homogenized in phosphate buffer 50mMpH 70 centrifuged at 10000timesg and the supernatant wasused for all antioxidant assays

Lipid peroxidation was determined by the presence ofthiobarbituric acid reactive substances in samples of pancreasand lung homogenates by the method previously describedby Bose et al [14] with minor modifications [10] The resultswere expressed as pmol of malondialdehyde (MDA) formedper mg of protein

Pancreatic and lung nonprotein sulfhydryl groups (NP-SH) were determined by Ellmanrsquos reaction using 5101584051015840-dithio-bis-2-nitrobenzoic acid as previously described [15] Resultswere expressed as 120583g of NP-SHmg of protein

The determination of oxidized protein content (carbonylgroups) was performed [16] by the reaction of carbonylgroups and 24-dinitrophenylhydrazine The concentrationof carbonyl groups was calculated by using 215mMminus1 cmminus1as the extinction coefficient for aliphatic hydrazones and theresults were expressed as nmol of carbonylsmg of protein

Tissue antioxidant status was evaluated in the homoge-nate of pancreas and lung tissues using the ferric reducingantioxidant power (FRAP) assay [17] Results were plottedagainst a standard curve of ferrous sulphate (500ndash1500120583molL) and were expressed as 120583mol of ferrous sulphatemg ofprotein

Catalase (CAT) activity was appraised by the decrease inabsorption of H2O2 at 240 nm due to H2O2 consumptionby CAT as previously described [18] Specific activity wasexpressed as units of CATmg of protein

The activity of CuZn SODwas assayed by the adrenalinemethod based on the capacity of SOD to inhibit autoxidationof adrenaline to adrenochrome One unit of SOD activity wasdefined as the amount of protein causing 50 inhibition ofthe autoxidation of adrenaline at 26∘C [19]

Glutathione peroxidase (GSH-Px) activity was deter-mined as described previously [20] Enzyme activity was


Inte

ns (

mAU

)

1 2 3

4

Cran_1-22_01_271d UV Chromatogram 270 nm

0

50

100

5 10 15 20 25 300Time (min)

(a)

Inte

ns (

mAU

)

5

67

05

101520


5 10 15 20 25 300Time (min)

(b)

Figure 1 Chromatograms obtained by HPLC-DAD at 120582 = 270 nm (a) and 525 nm (b) for the hydroethanolic extract of fruits of Vacciniummacrocarpon (HEVm) The identification of the peaks is given in Table 1

Table 1 Phenolic compounds identified in the hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) by HPLC-DAD-ESI-MSMS

Peak Proposed structure RT (min) Molecular ion MS2MS3 (mz) Concentrationlowastlowast

1 Catechinlowast 149 291 273 165 139 123 58182 plusmn 30502 Epicatechinlowast 185 291 273 165 139 123 9570 plusmn 4013 Quercetin-3-O-rutinoside 245 611 465 303 3789 plusmn 6554 Epicatechin gallatea 248 441 289 169 13922 plusmn 5075 Cyanidin-3-O-galactosideb 134 449 287 245 plusmn 0456 Cyanidin-3-O-glucosidelowast 144 449 287 1187 plusmn 0757 Cyanidin-coumaroyl-hexoside 147 595 449287 669 plusmn 054RT retention time Peaks are numbered according to Figures 1(a) and 1(b) lowastCompoundsrsquo identity was confirmed by comparison with RT of standards andmass fragment profile and UV-Vis absorption spectra lowastlowastExpressed as mean plusmn SD of mg100 g of extract aQuantified as equivalent of epicatechin bQuantifiedas equivalent of cyanidin-3-O-glucoside Compounds 1ndash3 and 4ndash7 were analyzed in positive mode compound 4 was analyzed in negative mode

expressed as 120583mol of glutathione oxidizedmg of pro-teinmin by using the extinction coefficient for NADPH(6220Mminus1 cmminus1)

211 Statistical Analysis Results were expressed as mean plusmnstandard error of mean (SEM) and analyzed by one- or two-way analysis of variance (ANOVA) followed by Bonferronirsquospost hoc test using the GraphPad Prism software (version50) 119901 lt 005 was considered significant

3 Results

31 Phytochemical Analysis of HEVm Figure 1 shows theHPLC-DAD chromatograms of the HEVm extract andthe respective identification of the compounds by LC-ESI-MSMS is shown in Table 1 Seven compoundswere identified(three flavan-3-ols one flavonol and three anthocyanins)The major compounds were catechin and epicatechin (Fig-ure 1(a) peaks 1 and 2 119898119911 291) The main anthocyaninswere cyanidin-3-O-glucoside (119898119911 449 peak 6 Figure 1(b))followed by cyanidin-coumaroyl-hexoside (119898119911 595 peak7 Figure 1(b)) both with characteristic molecular ions ofcyanidin (119898119911 287) The total flavonoids content in pow-dered cranberry was 87565mg100 g where flavan-3-ol con-tributed 933 followed by flavonol (43) and anthocyanins(24)

32 Effect of HEVm on Inflammatory Parameters Pancreaticinflammation induced by L-arginine was characterized by amarked increase of pancreatic MPO activity after 72 h Thetreatment with HEVm at 200mgkg prevented this effect(119901 lt 0001) (Figure 2(a)) In addition the inductionof pancreatitis significantly increased MPO activity in thelung tissue compared with the saline group This effect wasalso reduced by the treatment with HEVm at 200mgkg(119901 lt 0001 Figure 2(b)) L-arginine administration enhancedpancreatic edema index after injection (515 plusmn 005 119901 lt0001) compared with the saline group (439 plusmn 008) and thetreatment with HEVm at 200mgkg produced a significantinhibition of this effect (438 plusmn 008 119901 lt 0001) as diddexamethasone (438 plusmn 009 119901 lt 0001) However thetreatment with HEVm at 50 or 100mgkg did not alter edema(514 plusmn 014 492 plusmn 014 resp)

In addition to the increase of the above-mentionedinflammatory parameters the levels of TNF-120572 IL-1120573 andIL-6 were significantly increased in pancreas and lung afterAP induction by L-arginine (Figures 3(a)ndash3(f)) comparedwith animals injected with saline The treatment with HEVmstrongly modulated the levels of these cytokines Levels ofTNF-120572 were significantly decreased by the treatment withHEVm in the pancreatic (119901 lt 001 and 119901 lt 005 respat 100 and 200mgkg Figure 3(a)) and lung tissue (119901 lt0001 at 50 100 and 200mgkg Figure 3(b)) compared


UM

POm

g of

pan

crea

tic ti

ssue

Pancreas

0

5

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15

HEVm (mgkg)

AP

VehicleVehicle +Saline

50 100 200 Dexa(5 mgkg)

lowast

lowastlowastlowast

(a)

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AP

UM

POm

g of

lung

tiss

ue

Lung

0

3

6

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15


50 100 200 Dexa(5 mgkg)

lowastlowastlowast lowast

(b)

Figure 2 Effect of the hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) on pancreatic and lung myeloperoxidase (MPO)activityMicewere submitted to injection of L-arginine or saline treatedwithHEVm (50 100 or 200mgkg) dexamethasone (Dexa 5mgkg)or vehicle (saline) and euthanized after pancreatitis induction MPO activity was measured in pancreas (a) and lung (b) tissues (119899 = 5-6)119901 lt 0001 versus vehicle + saline and lowast119901 lt 005 or lowastlowastlowast119901 lt 0001 versus vehicle + L-arginine One-way ANOVA followed by Bonferronirsquostest

with vehicle-treated animals The amount of IL-1120573 was alsolower in pancreas (119901 lt 001 at 50 and 200mgkg and119901 lt 0001 at 100mgkg Figure 3(c)) and lung tissues(119901 lt 0001 Figure 3(d)) in the group treated with HEVmcompared to the vehicle-treated animals A similar effect wasobserved for IL-6 levels for which the groups treated withHEVm presented significantly lower levels of this cytokinein pancreas (119901 lt 0001 Figure 3(e)) and lung (119901 lt0001 Figure 3(f)) compared with vehicle-treated animalsAs a control treatment with dexamethasone decreased thelevels of these cytokines in both pancreas and lungs tissues(Figure 3)

33 Effect of HEVm on Mechanical Abdominal Hyperalge-sia L-arginine-induced AP was accompanied by a markedreduction of the intensity of stimulus needed to causewithdrawal behavior in mice after 48 or 72 h denoting thatmice developed abdominal hyperalgesia as a consequence ofthe pancreatic inflammation Two-way ANOVA indicated asignificant interaction between the time and treatment in thisexperiment (119901 lt 0001 F = 3712) The injection of salineinstead of L-arginine did not alter the micersquos withdrawalbehavior (Figure 4) in comparison to basal evaluation

Figure 4 also demonstrates that the treatmentwithHEVmclearly reduced the abdominal hyperalgesia of mice submit-ted to AP after 48 h (119901 lt 001 at 50mgkg and 119901 lt 005at 100mgkg) or 72 h (119901 lt 0001 at 50mgkg 119901 lt 001 at100mgkg and 119901 lt 005 at 200mgkg)

As an experimental control treatment with morphineinhibited abdominal hyperalgesia stimulus at 24 (97 plusmn 1 g119901 lt 0001) 48 (83 plusmn 2 g 119901 lt 0001) and 72 h (58 plusmn 2 g119901 lt 0001) after induction of pancreatitis compared withanimals treated with the vehicle (minus37 plusmn 1 minus81 plusmn 1 andminus101 plusmn 1 g resp at 24 48 and 72 h after AP)

Of interest no difference was observed in the mean basalvalues of intensity of stimulus needed to induce withdrawalbehavior among the groups (saline + vehicle 259 plusmn 20 gL-arginine + vehicle 282 plusmn 07 g L-arginine + HEVm[50mgkg] 265 plusmn 10 g L-arginine + HEVm [100mgkg]261 plusmn 03 g L-arginine + HEVm [200mgkg] 280 plusmn 08 gand L-arginine + morphine 271 plusmn 06 g)

34 Lack of Effect of HEVm on the Locomotor ActivityPretreatment of mice (119899 = 5) with HEVm (200mgkg) didnot modify the distance traveled (343plusmn31m) mobility time(258 plusmn 17 s) or the number of lines crossed (238 plusmn 17) whencompared to the vehicle group (363 plusmn 41m 275 plusmn 9 s and245 plusmn 20 resp) As a control pretreatment with diazepandecreased these parameters significantly (84plusmn36m 17plusmn5 sand 13 plusmn 3 119901 lt 0001 each) in comparison to the vehicle-treated group

35 Effect of HEVm on Serum Biochemical Parameters L-arginine-induced AP significantly increased the serum levelsof amylase and lipase This effect was accompanied byincrease of the serum concentrations of ALT and AST afterthe induction of AP compared with saline-injected animalsTreatment with HEVm reduced the serum levels of amylase(200mgkg 119901 lt 0001) lipase (200mgkg 119901 lt 001) ALT(100mgkg 119901 lt 005 200mgkg 119901 lt 005) and AST(100mgkg 119901 lt 005 200mgkg 119901 lt 001) Treatment withdexamethasone (5mgkg) decreased the levels of amylase(119901 lt 001) and lipase (119901 lt 005) in serum (Table 2)

36 Effect of HEVm on Oxidative Stress Markers and Antiox-idant Enzymes Lipid peroxidation products in particularMDA were higher in both pancreas (Figure 5(a)) and lung(Figure 5(b)) after the induction of pancreatitis This effect


TNF-

(p

gm

g of

pan

crea

tic p

rote

in) Pancreas

lowastlowastlowastlowast

0

50

100

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1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(a)

TNF-

(p

gm

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lung

pro

tein

)

Lung

lowastlowastlowast

lowastlowastlowast lowastlowastlowast lowastlowastlowast

0

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50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(b)

IL-1

(pg

mg

of p

ancr

eatic

pro

tein

) Pancreas

lowastlowast lowastlowast lowastlowastlowastlowastlowast

0

50

100

150400

600

800


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(c)

IL-1

(pg

mg

of lu

ng p

rote

in)

Lung

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

0

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50 100 200 DexaHEVm (mgkg)

AP

(5 mgkg)

(d)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

01020304050

100

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IL-6

(pg

mg

of p

ancr

eatic

pro

tein

)

Pancreas



AP

(5 mgkg)

(e)

lowastlowastlowast


0

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IL-6

(pg

mg

of lu

ng p

rote

in)

Lung


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(f)

Figure 3The hydroethanolic extract of fruits ofVacciniummacrocarpon (HEVm) inhibits tumor necrosis factor- (TNF-) 120572 interleukin- (IL-)1120573 and IL-6 levels in pancreas and lung Mice were submitted to injection of L-arginine or saline treated with HEVm (50 100 or 200mgkg)dexamethasone (Dexa 5mgkg) or vehicle (saline) and euthanized after pancreatitis induction TNF-120572 IL-1120573 and IL-6 levels weremeasuredin pancreas ((a) (c) and (e) resp) and lung ((b) (d) and (f) resp) tissues (119899 = 5-6) 119901 lt 005 or 119901 lt 0001 versus vehicle + saline andlowast119901 lt 005 lowastlowast119901 lt 001 or lowastlowastlowast119901 lt 0001 versus L-arginine + vehicle One-way ANOVA followed by Bonferronirsquos test

was inhibited by the treatment with HEVm at doses of 50100 and 200mgkg (119901 lt 0001 Figure 5(a)) in pancreatictissue comparedwith animals treatedwith the vehicle In lungtissue the production of MDA was also reduced by the dosesof 50 (119901 lt 005) 100 and 200mgkg (119901 lt 0001 Figure 5(b))compared with animals treated with the vehicle

In the pancreas and lung tissue of animals with AP higheramounts of carbonyl radicals were found (Figures 5(c) and5(d)) This effect was reversed in pancreas (119901 lt 005 at50mgkg and 119901 lt 001 at 100 and 200mgkg Figure 5(c))and lung (119901 lt 0001 for all doses Figure 5(d)) of animalstreated with HEVm


lowast

lowastlowastlowast

minus12

minus9

minus6

minus3

0

3

Δ of

with

draw

al th

resh

old

(g)

24 48 720Time (h)

Saline + VehicleL-Arginine + VehicleL-Arginine + HEVm (50 mgkg)

mgkg)L-Arginine + HEVm (200L-Arginine + HEVm (100 mgkg)

Figure 4 The hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) reduces abdominal hyperalgesia in L-arginine-inducedpancreatitis Animals were submitted to injection of L-arginine or saline treated with HEVm (50 100 or 200mgkg) or vehicle (saline) andeuthanized after pancreatitis induction The variation (Δ) of the threshold force to cause withdrawal behavior was calculated by subtractingeach time point from basal values (0 h) and was expressed in g for the intensity of stimulus for 119899 = 5 119901 lt 0001 versus the respective saline+ vehicle group lowast119901 lt 005 or lowastlowast119901 lt 001 versus the respective L-arginine + vehicle Two-way ANOVA (interaction of treatment versus time119901 lt 0001 and F(1581) = 3712) followed by Bonferronirsquos test

Table 2 The hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) reduces serum biochemical parameters in mice submittedto acute pancreatitis by L-arginine

Parameter Saline + vehicle Saline +L-arginine

HEVm (50mgkg)+ L-arginine

HEVm(100mgkg) +L-arginine


Dexa (5mgkg) +L-arginine

Amylase(UdL) 222 plusmn 21 968 plusmn 114a 873 plusmn 44 763 plusmn 73 346 plusmn 16e 458 plusmn 132d

Lipase (UdL) 69 plusmn 13 475 plusmn 102a 313 plusmn 23 370 plusmn 52 196 plusmn 26d 209 plusmn 26c

ALT (UIL) 42 plusmn 03 98 plusmn 03a 82 plusmn 12 63 plusmn 05c 56 plusmn 09c 72 plusmn 05AST (UIL) 64 plusmn 02 116 plusmn 15b 72 plusmn 07 61 plusmn 05e 71 plusmn 05d 85 plusmn 05Groups of mice (n = 5-6) were submitted to injection of L-arginine or saline treated with HEVm (50 100 or 200mgkg) dexamethasone (Dexa 5mgkg) orvehicle (saline) and serum was collected after 72 h of induction a119901 lt 001 and b119901 lt 005 versus the respective saline + vehicle group or c119901 lt 005 b119901 lt 001and e119901 lt 0001 versus the respective AP + vehicle group

In addition pancreatic and lung nonprotein sulfhydrylgroups (NP-SH) were decreased by AP induction (Figures5(e) and 5(f)) Treatment with HEVm at the doses of 100and 200mgkg maintained the NP-SH content in pancreas(119901 lt 005 Figure 5(e)) and lung (119901 lt 0001 Figure 5(f))compared with animals treated with the vehicle

Additionally tissue antioxidant status was measured inthe pancreas and lung through the determination of thetotal antioxidant activity by the reduction of iron (FRAP)Figures 5(g) and 5(h) demonstrate that tissue antioxidantcapacity was significantly reduced in pancreas and lung afterAP induction and the treatment with 200mgkg of HEVmmaintained this capacity near the levels observed in animals

from the vehicle + saline group in both pancreas (119901 lt 005Figure 5(g)) and lung (119901 lt 0001 Figure 5(h))

Since the treatment with HEVm produced a markedantioxidant effect in pancreas and lung tissue of animals withpancreatitis the contribution of some antioxidant enzymesto this action was studied The induction of AP reducedthe activity of CAT SOD and GSH-Px enzymes in pancreasand CAT and SOD activities in lung (Figure 6) Figure 6(a)shows the increase in pancreatic CAT activity of mice treatedwith HEVm at doses of 50mgkg (119901 lt 001) 100mgkg(119901 lt 0001) and 200mgkg (119901 lt 001) A similar effectwas observed in lungs (Figure 6(b)) of animals treated withHEVm at doses of 50 (119901 lt 005) 100 (119901 lt 001) and


Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)


50 100 200HEVm (mgkg)AP

lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)

Figure 5 Effect of the hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) on pancreatic and lung biomarkers of oxidativestress Animals were submitted to injection of L-arginine or saline treated with HEVm (50 100 or 200mgkg) or vehicle (saline) andeuthanized 72 h after pancreatitis induction Malondialdehyde (MDA) concentrations nonprotein sulfhydryl groups (NP-SH) carbonylgroups and ferric reducingantioxidant power (FRAP) were measured in pancreas ((a) (c) (e) and (g) resp) and lung ((b) (d) (f) and(h) resp) tissues (119899 = 5-6) 119901 lt 005 119901 lt 001 and 119901 lt 0001 versus vehicle + saline lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 versusvehicle + AP (one-way ANOVA followed by Bonferronirsquos test)



50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung

lowastlowast lowastlowast

0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)

Figure 6 Effect of the hydroethanolic extract of fruits of Vaccinium macrocarpon (HEVm) on activity of antioxidant enzymes in pancreasand lung Animals were submitted to injection of L-arginine or saline treated with HEVm (50 100 or 200mgkg) or vehicle (saline) andeuthanized 72 h after pancreatitis induction Catalase (CAT) superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity weremeasured in pancreas ((a) (c) and (e) resp) and lung ((b) (d) and (f) resp) tissues (119899 = 5-6) 119901 lt 005 119901 lt 001 and 119901 lt 0001versus vehicle + saline lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 versus vehicle + AP (one-way ANOVA followed by Bonferronirsquos test)

200mgkg (119901 lt 005) Concomitantly the activity of SODsignificantly increased in the pancreas after treatment withHEVm at doses of 50 (119901 lt 005) 100 (119901 lt 001) and200mgkg (119901 lt 0001 Figure 6(c)) which was also observed

in lung tissue after treatment with 100 (119901 lt 001) and200mgkg (119901 lt 005 Figure 6(d))However GPx activitywasnot modified by the treatment with HEVm in both pancreasand lung tissues (Figures 6(e) and 6(f))


4 Discussion

The need for new experimental alternatives to treat AP isstill a challenge since there are no specific treatments forclinical management of this disease [3] In this study wepresent data that demonstrates that HEVm can modulatethe inflammatory response in pancreas and lung (with theparticipation of oxidative stress) and abdominal hyperalgesiaduring L-arginine-induced AP The finding that HEVm hasbeneficial effect in experimental pancreatitis is novel forthis extract which is frequently used by people to treatinflammatory conditions of the urinary system [8] Howeverthe presence of flavonoids in this extract supports the pos-sibility of a beneficial effect on AP For this reason we usedthe hydroethanolic extract of cranberry Our current datareinforce this hypothesis in themodel of pancreatitis inducedby L-arginine

The phytochemical analysis of HEVm showed that cat-echins and epicatechins are its major components It waspossible to identify three flavan-3-ols one flavonol andthree anthocyanins Catechin epicatechins and cyanidinderivatives were previously described in cranberry prepara-tions [21 22] as was quercetin-3-O-rutinoside (rutin) [23]These compounds exert various biological effects includingantioxidant and anti-inflammatory effects [11 21]

Treatment with HEVm induced protective effects againstpancreatic inflammation The activity of MPO edema indexand production of cytokines (TNF-120572 IL-1120573 and IL-6) werereduced by this extract Along with the lower pancreaticdamage in animals treated with HEVm the levels of serumamylase and lipase were lower which corroborates thereduction of pancreatic damage Neutrophil infiltration isa hallmark of AP [24] and our data show that HEVm caninterfere with the presence of these cells in the pancreasThisagrees with previous findings that cranberry extract reducesthe MPO activity in mice paws injected with carrageenan[23] Similarly treatment with 15 dried whole cranberrypowder (incorporated in the diet) reduced MPO activity inthe colon of mice submitted to dextran sulphate sodium-induced colitis [25] Altogether these results highlight theanti-inflammatory effect of the extract of fruits of V macro-carpon In accordance with this effect treatment with HEVmproduced antiedematogenic effect Corroborating this find-ing [23] showed that the extract of V macrocarpon fruitsreduces the production of prostaglandins in vitro possessesantioxidant activity and inhibits carrageenan-induced micepaw edema The reduction of neutrophil migration to thepancreas and edema formation induced by HEVm certainlyare dependent on the inhibitory activity of proinflammatorycytokines Our results demonstrate that pancreatic contentsof TNF-120572 IL-1120573 and IL-6were reduced by the treatmentwithHEVmOther studies have also demonstrated that the extractof cranberry inhibited IL-6 IL-8 and prostaglandin E2 pro-duction by gingival fibroblasts stimulated with lipopolysac-charide in vitro [26] and that serum IL-1120573 and interferon-120574 concentrations and TNF-120572 and IL-1120573 mRNA expressionin colon were reduced by treatment with cranberry inmice submitted to dextran sulphate sodium-induced colitis[25]

Besides pancreatic inflammation abdominal hyperalge-sia was observed 48 and 72 hours after pancreatitis inductionSimilar results were described by Abreu et al [11] in themodel of L-arginine-induced pancreatitis and we believethat this mimics the abdominal pain observed in humansafter an AP attack The reduction of AP-induced abdom-inal hyperalgesia by the treatment with HEVm may be aconsequence of the decrease of pancreatic inflammatorymediators such as TNF-120572 IL-1120573 and IL-6 However wecannot discard the possibility that substances present inHEVm blocked the stimulation of nociceptive pathwaysSince HEVm administration did not alter the locomotoractivity of mice we can exclude the possibility of muscularrelaxation or central nervous system depression that couldbias the nociceptive measurement Few studies have focusedon the possible analgesic effect of V macrocarpon fruits Arandomized clinical study demonstrated that men receivingradiation therapy for prostate cancer and taking cranberrycapsules presented lower incidence of pain and burning[27] The presence of flavonoids could also contribute tothis antihyperalgesic effect A previous study showed thatthe extract of grapefruit rich in proanthocyanidin (a classof flavonoids) reduced nociception caused by sciatic nerveligation-induced neuropathic pain in rats [28] In additionthe grapefruit extract reduced the nociception induced byformalin in mice by mechanisms involving the participationof opioid receptor activation [29] Concordantly a previousstudy by our group showed that rutin reduced abdominalhyperalgesia in L-arginine-induced pancreatitis in mice [11]

Following AP induction many extrapancreatic responseswere observed in animals injected with L-arginine such asthe increased MPO activity in lung and increased markersof hepatic injury Although the mechanisms underlyingthese alterations are not well understood in this modelof pancreatitis the beneficial effect of HEVm is supportedby these results Serum ALT and AST levels were used asmarkers of extrapancreatic alterations in the present studyThe increase in ALT but not AST is usually predictiveof biliary pancreatitis [30] However in the model of L-arginine-induced AP in rats other authors have shown thatboth AST and ALT are increased [31] but no study haspreviously measured these enzymes in mice Interestinglythis increase in hepatic enzymes was partially reduced by theadministration of HEVm which may in part be due to thereduction of pancreatic lesions as observed by the decreaseof pancreatic inflammatory markers and enzymes (amylaseand lipase)

Lung injury is the secondary effect of pancreatitismost related to mortality in patients [32] In the modelof L-arginine-induced pancreatitis lung MPO activity wasincreased 72 h after induction corroborating previous find-ings [9] The fact that the treatment with HEVm reducedlungMPOactivity is complementary to the above-mentionedextrapancreatic protective effects which might be a conse-quence of the reduction of pancreatic damage Pancreatitis-related lung injury is multifactorial and is associated withmany factors such as the presence of activated enzymesdestruction of the lung surfactant low lung perfusion ratesand increased cytokine in lung tissue [33] Interestingly


in agreement with the decrease in lung MPO activity wedetected lower TNF-120572 IL-1120573 and IL-6 levels in lungs ofanimals treated with HEVm in comparison to the vehicle-treated group

Since theHEVm is rich in flavanols and other polyphenolsand these compounds have antioxidant effects [11 34 35] wespeculated that the beneficial actions observed in pancreaticand extrapancreatic parameters could take place with theinvolvement of antioxidant activity In fact increased TBARSand carbonyl groups in parallel with decreased NP-SH andtotal antioxidant capacity (FRAP) in both pancreas and lungindicated that oxidative stress plays a significant role inthis model as previously shown [11 36] Treatment withHEVm maintained antioxidant capacity of the tissue (NP-SH and FRAP) and consequently reduced the oxidationmarkers (MDA and carbonyl) which can be attributedto the direct antioxidant effect of polyphenols found inHEVm [11 22 23 34 37] Another reasonable possibility ofexplaining the antioxidant effect of HEVm is modulation ofthe activity of endogenous antioxidant enzymes Pancreatitisinduction was characterized by reduction of the activity ofCAT SOD and GPx in pancreas and lung corroboratingprevious findings [11] The CAT and SOD activities weremarkedly altered by the treatment with HEVm indicatingthat HEVm strongly modulates oxidative stress InterestinglyCAT and SOD activities seemed to be more susceptible toreversion in pancreas than in lung which agrees with theidea that the reduction of pancreatic damage was importantto the modulation of extrapancreatic effects The beneficialmodulation of CAT and SOD activities in animals treatedwith HEVm was more evident than GPx activity whichwas not altered by treatment with HEVm Indeed HEVmcountered the L-arginine-induced increase in oxidation anddecrease in antioxidative capacity

The antioxidant effects observed in the treatment withHEVm can be caused by the compounds present in thisextract The phenolic compounds found in HEVm likeanthocyanins rutin catechin and epicatechins and otherphenolic compounds (eg quercetin kaempferol ellagicacid and p-coumaric acid) are present in cranberries (forreview see [21 22]) and certainly contributed to the reductionof oxidative stress which in turn decreased the pancreaticlesions and resulted in less severe extrapancreatic alterationsin mice

An important concern about cranberry extracts is regard-ing their safety Many studies have found no toxicity aftertreatment with cranberry extracts for 6ndash10 weeks in exper-imental animals [5 6 38] or in humans [39 40] but arecent study found small differences in the spleen and kidneyfunction of the progeny ofmice treatedwith cranberry extractduring pregnancy [41]Thusmore specific studies are neededto assure the safety of cranberry extracts

Altogether our results indicate a protective role of HEVmin the model of AP induced by L-arginine by reducing pan-creatic damage hyperalgesia and remote alterations throughthe key participation of the antioxidant capacity of thisextract These findings provide insights into the possible useof this extract in the treatment of this disease

Abbreviations

ALT Alanine aminotransferaseAP Acute pancreatitisAST Aspartate aminotransferaseCAT CatalaseELISA Enzyme-linked immunosorbent assayESI Electrospray ionizationFRAP Ferric reducingantioxidant powerGPx Glutathione peroxidaseHEVm Hydroethanolic extract of Vaccinium

macrocarpon

HPLC-DAD High-performance liquidchromatography coupled to the detectordiode array

IL InterleukinMDA MalondialdehydeMPO MyeloperoxidaseNP-SH Nonprotein sulfhydryl groupsROS Reactive oxygen speciesSOD Superoxide dismutaseTNF-120572 Tumor necrosis factor-120572

Data Availability

The data used to support the findings of this study can beaccessed upon request to the corresponding author

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

The authors acknowledge ldquoConselho Nacional de Pesquisae Desenvolvimento Cientıficordquo (CNPq) and Fundacaode Amparo a Pesquisa do Estado de Sao Paulo (Grant2013079914-8) for financial support Enilton AparecidoCamargo is beneficiary of a CNPq productivity grant

References

[1] S E Roberts K Thorne P A Evans A Akbari D G Samueland J G Williams ldquoMortality following acute pancreatitisSocial deprivation hospital size and time of admission Recordlinkage studyrdquo BMC Gastroenterology vol 14 no 1 article no153 2014

[2] M Nesvaderani G D Eslick and M R Cox ldquoAcute pancreati-tis Update on managementrdquo Medical Journal of Australia vol202 no 8 pp 420ndash423 2015

[3] M Yokoe T Takada and T Mayumi ldquoJapanese guidelinesfor the management of acute pancreatitis Japanese Guidelines2015rdquo Journal of Hepato-Biliary-Pancreatic Sciences vol 22 no6 pp 405ndash432 2015

[4] S Perez J Pereda L Sabater and J Sastre ldquoRedox signaling inacute pancreatitisrdquo Redox Biology vol 5 pp 1ndash14 2015

[5] F F Anhe D Roy and G Pilon ldquoA polyphenol-rich cranberryextract protects from diet-induced obesity insulin resistanceand intestinal inflammation in association with increased


Akkermansia spp population in the gut microbiota of micerdquoGut vol 64 pp 872ndash883 2014

[6] S L Glisan C Ryan A P Neilson and J D LambertldquoCranberry extract attenuates hepatic inflammation in high-fat-fed obese micerdquoThe Journal of Nutritional Biochemistry vol 37pp 60ndash66 2016

[7] I Singh L K Gautam and I R Kaur ldquoEffect of oral cranberryextract (standardized proanthocyanidin-A) in patients withrecurrent UTI by pathogenic E coli a randomized placebo-controlled clinical research studyrdquo International Urology andNephrology vol 48 no 9 pp 1379ndash1386 2016

[8] J B Blumberg T A Camesano A Cassidy et al ldquoCranberriesand their bioactive constituents in human healthrdquo Advances inNutrition vol 4 no 6 pp 618ndash632 2013

[9] R Dawra R Sharif P Phillips V Dudeja D Dhaulakhandiand A K Saluja ldquoDevelopment of a new mouse model of acutepancreatitis induced by administration of L-argininerdquo Ameri-can Journal of Physiology-Gastrointestinal and Liver Physiologyvol 292 no 4 pp G1009ndashG1018 2007

[10] D G Santana C A Santos A D C Santos et al ldquoBeneficialeffects of the ethanol extract of Caesalpinia pyramidalis on theinflammatory response and abdominal hyperalgesia in rats withacute pancreatitisrdquo Journal of Ethnopharmacology vol 142 no2 pp 445ndash455 2012

[11] F F Abreu A C A Souza S A Teixeira et al ldquoElucidatingthe role of oxidative stress in the therapeutic effect of rutin onexperimental acute pancreatitisrdquo Free Radical Research vol 50no 12 pp 1350ndash1360 2016

[12] F R Capaz L E Vanconcellos S De Moraes and J P NetoldquoThe open field a simple method to show ethanol withdrawalsymptomsrdquo Arch Int Pharmacodyn Ther vol 251 pp 228-361981

[13] P P Bradley D A Priebat R D Christensen and G RothsteinldquoMeasurement of cutaneous inflammation estimation of neu-trophil content with an enzyme markerrdquo Journal of InvestigativeDermatology vol 78 no 3 pp 206ndash209 1982

[14] R Bose G R Sutherland and C Pinsky ldquoBiological andmethodological implications of prostaglandin involvement inmouse brain lipid peroxidation measurementsrdquo NeurochemicalResearch vol 14 no 3 pp 217ndash220 1989

[15] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 pp 192ndash205 1968

[16] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[17] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of rsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[18] P Fossati L Prencipe and G Berti ldquoUse of 35-dichloro-2-hydroxybenzenesulfonic acid4-aminophenazonechromogenic system in direct enzymic assay of uric acidin serum and urinerdquo Clinical Chemistry vol 26 no 2 pp227ndash231 1980

[19] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquoThe Journal of Biological Chemistry vol 247 no 10pp 3170ndash3175 1972

[20] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo Journal of Laboratory and Clinical Medicine vol 70no 1 pp 158ndash169 1967

[21] E Pappas and K M Schaich ldquoPhytochemicals of cranberriesand cranberry products Characterization potential healtheffects and processing stabilityrdquoCritical Reviews in Food Scienceand Nutrition vol 49 no 9 pp 741ndash781 2009

[22] S Skrovankova D Sumczynski J Mlcek T Jurikova andJ Sochor ldquoBioactive compounds and antioxidant activity indifferent types of berriesrdquo International Journal of MolecularSciences vol 16 no 10 pp 24673ndash24706 2015

[23] G M Nardi A G De Farias Januario C G Freire et alldquoAnti-inflammatory activity of berry fruits in mice modelof inflammation is based on oxidative stress modulationrdquoPharmacognosy Research vol 8 pp S42ndashS49 2016

[24] P Szatmary and I Gukovsky ldquoThe Role of Cytokines andInflammation in the Genesis of Experimental PancreatitisrdquoPancreapedia Exocrine Pancreas Knowledge Base vol 29 2016

[25] X Xiao J Kim Q Sun et al ldquoPreventive effects of cranberryproducts on experimental colitis induced by dextran sulphatesodium in micerdquo Food Chemistry vol 167 pp 438ndash446 2015

[26] D A Tipton A A Hatten J P Babu and M K DabbousldquoEffect of glycated albumin and cranberry components oninterleukin-6 and matrix metalloproteinase-3 production byhuman gingival fibroblastsrdquo Journal of Periodontal Research vol51 no 2 pp 228ndash236 2016

[27] K Hamilton N C Bennett G Purdie and P M Herst ldquoStan-dardized cranberry capsules for radiation cystitis in prostatecancer patients in New Zealand a randomized double blindedplacebo controlled pilot studyrdquo Supportive Care in Cancer vol23 no 1 pp 95ndash102 2015

[28] G Kaur O Bedi N Sharma S Singh R Deshmukh and PKumar ldquoAnti-hyperalgesic and anti-nociceptive potentials ofstandardized grape seed proanthocyanidin extract against CCI-induced neuropathic pain in ratsrdquo Journal of Basic and ClinicalPhysiology and Pharmacology vol 27 no 1 pp 9ndash17 2016

[29] S Uchida K Hirai J Hatanaka J Hanato K Umegaki andS Yamada ldquoAntinociceptive effects of St Johnrsquos wort Harpago-phytum procumbens extract and grape seed proanthocyanidinsextract in micerdquo Biological amp Pharmaceutical Bulletin vol 31no 2 pp 240ndash245 2008

[30] N O Zarnescu R Costea E C Zarnescu Vasiliu and S NeaguldquoClinico-biochemical factors to early predict biliary etiologyof acute pancreatitis age female gender and ALTrdquo Journal ofMedicine and Life vol 8 no 4 pp 523ndash526 2015

[31] A Yenicerioglu Z Cetinkaya M Girgin et al ldquoEffects oftrimetazidine in acute pancreatitis induced by L-argininerdquoCanadian Journal of Surgery vol 56 no 3 pp 175ndash179 2013

[32] R Pezzilli L Bellacosa and C Felicani ldquoLung injury in acutepancreatitisrdquo Journal of the Pancreas vol 10 no 5 pp 481ndash4842009

[33] M-T Zhou C-S Chen B-C Chen Q-Y Zhang and RAndersson ldquoAcute lung injury and ARDS in acute pancreatitismechanisms and potential interventionrdquo World Journal ofGastroenterology vol 16 no 17 pp 2094ndash2099 2010

[34] F-Y Fan L-X Sang M Jiang and D J McPhee ldquoCatechinsand their therapeutic benefits to inflammatory bowel diseaserdquoMolecules vol 22 no 3 article no 484 2017

[35] A S Kristo D Klimis-Zacas and A K Sikalidis ldquoProtectiverole of dietary berries in cancerrdquo Antioxidants vol 5 no 4article no 37 2016

[36] C M Melo K M M B Carvalho J C de Sousa Neves etal ldquo120572120573-amyrin a natural triterpenoid ameliorates L-arginine-induced acute pancreatitis in ratsrdquo World Journal of Gastroen-terology vol 16 no 34 pp 4272ndash4280 2010


[37] S Zafra-Stone T YasminM Bagchi AChatterjee J AVinsonand D Bagchi ldquoBerry anthocyanins as novel antioxidants inhuman health and disease preventionrdquo Molecular Nutrition ampFood Research vol 51 no 6 pp 675ndash683 2007

[38] J K Prasain K Jones R Moore et al ldquoEffect of cranberry juiceconcentrate on chemically-induced urinary bladder cancersrdquoOncology Reports vol 19 no 6 pp 1565ndash1570 2008

[39] J-J Dugoua D Seely D Perri E Mills and G KorenldquoSafety and efficacy of cranberry (Vaccinium Macrocarpon)during pregnancy and lactationrdquo Canadian Journal of ClinicalPharmacology vol 15 no 1 pp e80ndashe86 2008

[40] A Ledda G Belcaro M Dugall et al ldquoHighly standardizedcranberry extract supplementation (Anthocran) as prophy-laxis in young healthy subjects with recurrent urinary tractinfectionsrdquo European Review for Medical and PharmacologicalSciences vol 21 no 2 pp 389ndash393 2017

[41] B J Bałan S Lewicki A K Siwicki et al ldquoMorphometricabnormalities in spleen and kidney of the progeny of mice fedAmerican cranberry extract (Vaccinium macrocarpon) duringpregnancy and lactationrdquo Polish Journal of Veterinary Sciencevol 20 no 1 pp 57ndash65 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018


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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


of reactive oxygen species (ROS) and inflammatory cellinfiltration in the pancreas play a key role in the developmentof AP and complications associated with this condition [4]

The fruit ofVacciniummacrocarpon Aiton (Ericaceae) ispopularly known as cranberry and is used to make juices andsauces throughout North America Due to promising resultsthe medicinal use of this plant has spread throughout tothe Western world [5] It contains anthocyanins flavonoidsproanthocyanidins condensed tannins and phenolic acidsRecent studies have shown that an extract of fruits of Vmacrocarpon containing approximately 182 anthocyaninscan attenuate hepatic inflammation in obese mice [6] andinhibit the adhesion of pathogenic E coli in patients withrecurrent urinary tract infection [7] In addition treatmentwith this extract led to decreased intestinal inflammation inobese mice [5] Clinically cranberry preparations have beenused in the treatment of urinary tract diseases and vascularinflammation demonstrating high therapeutic potential [8]which may still be underestimated

The need for new therapeutic options to treat AP alongwith the potential anti-inflammatory and antioxidant effectpresented by cranberry makes it a viable alternative for thetreatment pancreatitis Thus we investigated the possibleeffect of a hydroethanolic extract of fruits of Vacciniummacrocarpon (HEVm) in experimental AP in mice

2 Material and Methods

21 Plant Material The hydroethanolic extract of fruits ofVacciniummacrocarpon (HEVm)was purchased fromFagronBV lot number 14083757D with certificate of quality controlanalysis (HHHC140402)

22 Characterization of Hydroethanolic Extract of Fruitsof Vaccinium macrocarpon Samples were dissolved withmethanol water (70 30 v v) acidified with 5 acetic acidusing an ultrasound bath (Ultrasonic Cleaner 8892E-DTHCole-Parmer Illinois USA) After 10min at room temper-ature the dissolved extract was centrifuged at 3500timesg for10min at 4∘C This procedure was repeated 3 times and thesupernatants were combinedThe supernatants were reducedunder vacuum at 40∘C in a rotary evaporator (RotavaporRE 120 Buchi Flawil Switzerland) prior to passage througha C-18 extraction column (1 g) (Supelclean LC-18 SupelcoPennsylvania USA) previously conditioned with methanoland ultrapure water The sample was loaded into the columnand washed with ultrapure water The phenolic compoundswere eluted with methanol acidified with 03 HCl Theeluateswere completely dried using a rotary evaporator undervacuum at 40∘C resuspended in methanol acidified with5 acetic acid and filtered through a 045 120583m PVDF filter(Millipore Ltd) for the quantification of flavonoids by high-performance liquid chromatography-diode array detection(HPLC-DAD) analysis

Quantification of flavonoidswas carried out using anAgi-lent 1260 Infinity Quaternary LC System (Agilent Technolo-gies USA) coupled to a diode array detector (DAD)The col-umnusedwas a 5 120583mProdigyODS3 column (460times 250mm)(Phenomenex Ltd UK) with a flow rate of 1mLmin at 25∘C

The mobile phase consisted of waterformic acidacetonitrile(96 1 3 solvent A and 58 1 51 solvent B) The solventgradient was 10 B at the beginning 25 at 10 minutes31 at 15 minutes 40 at 20 minutes 50 at 30 minutes100 at 40 minutes 10 at 45 minutes and 10 at 50minutes Chromatograms were acquired at 270 370 and525 nm The flavonoids were identified by comparing theirretention times diode array spectral characteristics andmassspectra analyzed using LC-ESI-MSMS with the standardsand the data available in the literature The quantificationwas done using a calibration curve of the commercial stan-dards (catechin epicatechin from Sigma-Aldrich (St LouisMO USA) and cyanidin-3-O-glucoside from ExtrasyntheseGenay France)

The identification of the flavonoids was performed usinga Prominence Liquid Chromatograph (Shimadzu Japan)linked to an AmaZon SL ion trap mass spectrometer (BrukerDaltonics BremenGermany)with an electrospray ionization(ESI) interfaceThe solvent gradient conditionswere the sameas those used for the HPLC-DAD analysis After the samplewas passed through the DAD the flow rate was changedto 08mLmin for examination by the mass spectrometerThe ESI was used in the positive and negative mode Themass spectrometer operating condition was collision energyof 4500V for positive and negative mode The analysiswas carried out using a full scan from 119898119911 100 to 1500The compounds were identified according to mass spectralcharacteristics and comparison with literature data

23 Animals Male adult Swiss mice (30ndash35 g) were obtainedfrom the Animal Center of Federal University of SergipeThe animals were maintained at 21 plusmn 2∘C with free access tofood (Nuvilab) and filtered water under a 12 12 h lightdarkcycle All experimental procedures were approved by theEthics Committee for Animal Use in Research of FederalUniversity of Sergipe (protocol number 0916) and wereconducted in compliance with the Guide for Care and Use ofLaboratory Animals (National Institutes of Health) Duringthe experiments animals were randomly distributed amongthe experimental groups and the experimenter was unawareof each grouprsquos identification

24 Experimental Procedure Pancreatitis induction was per-formed by two intraperitoneal (ip) injections 1 h apartof L- arginine at the dose of 4 gkg [9] The solution forinjection contained 8 L-arginine in sterile saline with pHadjusted to 70 The control group received sterile saline(NaCl 09) Seventy-two hours after the last injectionthe animals were anesthetized (80mgkg of ketamine plus10mgkg of xylazine) euthanized by exsanguination andtranscardially perfused with saline plus heparin (5 IUL) andtissue samples (pancreas and lung) were collected Blood wascentrifuged at 1000timesg for 15min at room temperature toseparate serum samples Aliquots of serum and tissues werestored at minus80∘C for proper analysis

25 Experimental Design Animals were randomly dis-tributed into five groups (119899 = 5-6 each) In the saline +vehicle group animals were injected with sterile saline (09





















Inte

ns (

mAU

)

1 2 3

4


0

50

100

5 10 15 20 25 300Time (min)

(a)

Inte

ns (

mAU

)

5

67

05

101520


5 10 15 20 25 300Time (min)

(b)







3 Results





UM

POm

g of

pan

crea

tic ti

ssue

Pancreas

0

5

10

15

HEVm (mgkg)

AP


50 100 200 Dexa(5 mgkg)

lowast

lowastlowastlowast

(a)

HEVm (mgkg)

AP

UM

POm

g of

lung

tiss

ue

Lung

0

3

6

9

12

15


50 100 200 Dexa(5 mgkg)


(b)











TNF-

(p

gm

g of

pan

crea

tic p

rote

in) Pancreas


0

50

100

150

200400

700

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(a)

TNF-

(p

gm

g of

lung

pro

tein

)

Lung

lowastlowastlowast


0

200

400

600

800

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(b)

IL-1

(pg

mg

of p

ancr

eatic

pro

tein

) Pancreas


0

50

100

150400

600

800


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(c)

IL-1

(pg

mg

of lu

ng p

rote

in)

Lung


lowastlowastlowast

0

200

400

600

800



AP

(5 mgkg)

(d)

lowastlowastlowast


01020304050

100

150

200

IL-6

(pg

mg

of p

ancr

eatic

pro

tein

)

Pancreas



AP

(5 mgkg)

(e)

lowastlowastlowast


0

50

100

150

200

IL-6

(pg

mg

of lu

ng p

rote

in)

Lung


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(f)





lowast

lowastlowastlowast

minus12

minus9

minus6

minus3

0

3

Δ of

with

draw

al th

resh

old

(g)

24 48 720Time (h)


















Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast


lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)



lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast


0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)




50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of







































Inte

ns (

mAU

)

1 2 3

4


0

50

100

5 10 15 20 25 300Time (min)

(a)

Inte

ns (

mAU

)

5

67

05

101520


5 10 15 20 25 300Time (min)

(b)







3 Results





UM

POm

g of

pan

crea

tic ti

ssue

Pancreas

0

5

10

15

HEVm (mgkg)

AP


50 100 200 Dexa(5 mgkg)

lowast

lowastlowastlowast

(a)

HEVm (mgkg)

AP

UM

POm

g of

lung

tiss

ue

Lung

0

3

6

9

12

15


50 100 200 Dexa(5 mgkg)


(b)











TNF-

(p

gm

g of

pan

crea

tic p

rote

in) Pancreas


0

50

100

150

200400

700

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(a)

TNF-

(p

gm

g of

lung

pro

tein

)

Lung

lowastlowastlowast


0

200

400

600

800

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(b)

IL-1

(pg

mg

of p

ancr

eatic

pro

tein

) Pancreas


0

50

100

150400

600

800


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(c)

IL-1

(pg

mg

of lu

ng p

rote

in)

Lung


lowastlowastlowast

0

200

400

600

800



AP

(5 mgkg)

(d)

lowastlowastlowast


01020304050

100

150

200

IL-6

(pg

mg

of p

ancr

eatic

pro

tein

)

Pancreas



AP

(5 mgkg)

(e)

lowastlowastlowast


0

50

100

150

200

IL-6

(pg

mg

of lu

ng p

rote

in)

Lung


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(f)





lowast

lowastlowastlowast

minus12

minus9

minus6

minus3

0

3

Δ of

with

draw

al th

resh

old

(g)

24 48 720Time (h)


















Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast


lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)



lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast


0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)




50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















UM

POm

g of

pan

crea

tic ti

ssue

Pancreas

0

5

10

15

HEVm (mgkg)

AP


50 100 200 Dexa(5 mgkg)

lowast

lowastlowastlowast

(a)

HEVm (mgkg)

AP

UM

POm

g of

lung

tiss

ue

Lung

0

3

6

9

12

15


50 100 200 Dexa(5 mgkg)


(b)











TNF-

(p

gm

g of

pan

crea

tic p

rote

in) Pancreas


0

50

100

150

200400

700

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(a)

TNF-

(p

gm

g of

lung

pro

tein

)

Lung

lowastlowastlowast


0

200

400

600

800

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(b)

IL-1

(pg

mg

of p

ancr

eatic

pro

tein

) Pancreas


0

50

100

150400

600

800


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(c)

IL-1

(pg

mg

of lu

ng p

rote

in)

Lung


lowastlowastlowast

0

200

400

600

800



AP

(5 mgkg)

(d)

lowastlowastlowast


01020304050

100

150

200

IL-6

(pg

mg

of p

ancr

eatic

pro

tein

)

Pancreas



AP

(5 mgkg)

(e)

lowastlowastlowast


0

50

100

150

200

IL-6

(pg

mg

of lu

ng p

rote

in)

Lung


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(f)





lowast

lowastlowastlowast

minus12

minus9

minus6

minus3

0

3

Δ of

with

draw

al th

resh

old

(g)

24 48 720Time (h)


















Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast


lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)



lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast


0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)




50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















TNF-

(p

gm

g of

pan

crea

tic p

rote

in) Pancreas


0

50

100

150

200400

700

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(a)

TNF-

(p

gm

g of

lung

pro

tein

)

Lung

lowastlowastlowast


0

200

400

600

800

1000


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(b)

IL-1

(pg

mg

of p

ancr

eatic

pro

tein

) Pancreas


0

50

100

150400

600

800


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(c)

IL-1

(pg

mg

of lu

ng p

rote

in)

Lung


lowastlowastlowast

0

200

400

600

800



AP

(5 mgkg)

(d)

lowastlowastlowast


01020304050

100

150

200

IL-6

(pg

mg

of p

ancr

eatic

pro

tein

)

Pancreas



AP

(5 mgkg)

(e)

lowastlowastlowast


0

50

100

150

200

IL-6

(pg

mg

of lu

ng p

rote

in)

Lung


50 100 200HEVm (mgkg)

AP

Dexa(5 mgkg)

(f)





lowast

lowastlowastlowast

minus12

minus9

minus6

minus3

0

3

Δ of

with

draw

al th

resh

old

(g)

24 48 720Time (h)


















Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast


lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)



lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast


0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)




50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















lowast

lowastlowastlowast

minus12

minus9

minus6

minus3

0

3

Δ of

with

draw

al th

resh

old

(g)

24 48 720Time (h)


















Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast


lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)



lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast


0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)




50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Pancreas

0

10

20

30M

DA

(pm

olm

g of

pan

crea

tic p

rote

in)


Lung

0

10

20

30

MD

A(p

mol

mg

of lu

ng p

rote

in)

lowast


lowastlowast

0

1

2

3

4

5

NP-

SH(

gm

g of

pan

crea

tic p

rote

in)


0

1

2

3

4

5

NP-

SH(

gm

g of

lung

pro

tein

)


lowast

00

05

10

154

7

10

Carb

onyl

(nm

olm

g of

pan

crea

tic p

rote

in)

(e) (f)

(c) (d)

(a) (b)



lowastlowastlowast

(h)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

lung

pro

tein

)



lowast

(g)

0

20

40

60

80

FRA

P (

mol

of

Fem

g of

pan

crea

tic p

rote

in)

lowastlowastlowast


0

2

4

6

8

10

Carb

onyl

(nm

olm

g of

lung

pro

tein

)




50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















50 100 200HEVm (mgkg)

AP

Pancreas


lowastlowastlowast

0

5

10

15

20

CAT

(Um

g of

pan

crea

tic p

rote

in)

(a)


50 100 200HEVm (mgkg)

AP

Lung


0

5

10

15

20

CAT

(Um

g of

lung

pro

tein

)

(b)


50 100 200HEVm (mgkg)

AP

lowast

lowastlowast

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

pan

crea

tic p

rote

in)

Pancreas

(c)


50 100 200HEVm (mgkg)

AP

lowastlowastlowast

0

5

10

15

20

SOD

(Um

g of

lung

pro

tein

)

Lung

(d)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

gof

pan

crea

tic p

rote

inm

in)

Pancreas

(e)


50 100 200HEVm (mgkg)

AP

0

10

20

30

GPx

(m

ol o

f GSS

Gm

g o

f lun

g pr

otei

nm

in)

Lung

(f)





4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















4 Discussion













Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























Abbreviations


macrocarpon



Data Availability




Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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